Variant icos ligand immunomodulatory proteins and related compositions and methods

ABSTRACT

Provided herein are immunomodulatory proteins comprising ICOSL variants and nucleic acids encoding such proteins. The immunomodulatory proteins provide therapeutic utility for a variety of immunological and oncological conditions. Compositions and methods for making and using such proteins are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional patentapplication 62/574,161, filed Oct. 18, 2017, entitled “VARIANT ICOSLIGAND IMMUNOMODULATORY PROTEINS AND RELATED COMPOSITIONS AND METHODS,”the contents of which are incorporated by reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled761612002240SeqList.txt, created Oct. 13, 2018, which is 1,655,996 bytesin size. The information in the electronic format of the SequenceListing is incorporated by reference in its entirety.

FIELD

The present disclosure relates to therapeutic compositions formodulating immune response in the treatment of cancer and immunologicaldiseases. In some aspects, the present disclosure relates to particularvariants of ICOS Ligand (ICOSL) that exhibit improved binding, such asimproved affinity or selectivity for one or both of the cognate bindingpartner proteins ICOS or CD28.

BACKGROUND

Modulation of the immune response by intervening in the processes thatoccur in the immunological synapse (IS) formed by and betweenantigen-presenting cells (APCs) or target cells and lymphocytes is ofincreasing medical interest. Mechanistically, cell surface proteins inthe IS can involve the coordinated and often simultaneous interaction ofmultiple protein targets with a single protein to which they bind. ISinteractions occur in close association with the junction of two cells,and a single protein in this structure can interact with both a proteinon the same cell (cis) as well as a protein on the associated cell(trans), likely at the same time. Although therapeutics are known thatcan modulate the IS, improved therapeutics are needed. Provided areimmunomodulatory proteins, including soluble proteins or transmembraneimmunomodulatory proteins capable of being expressed on cells, that meetsuch needs.

SUMMARY

Provided herein is a variant ICOS Ligand (ICOSL) polypeptide containingone or more amino acid modifications in an immunoglobulin superfamily(IgSF) domain of an ICOSL reference polypeptide, wherein the ICOSLreference polypeptide is a truncated extracellular domain comprising acontiguous sequence of amino acids comprising amino acids 1-112 and aC-terminal truncation of at least 25 amino acids with reference to theICOSL extracellular domain sequence set forth in SEQ ID NO: 32. In someof any of the provided embodiments, the variant ICOSL polypeptideexhibits altered binding to the ectodomain(s) of ICOS or CD28 comparedto the binding of the ICOSL reference polypeptide for the sameectodomain(s). In some of any of the provided embodiments, the variantICOSL polypeptide exhibits increased binding to the ectodomain(s) ofICOS or CD28 compared to the binding of the ICOSL reference polypeptidefor the same ectodomain(s).

In some of any such embodiments, the C-terminal truncation is of atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90, at least 100, at least 125 amino acid residues. In someof any of the provided embodiments, the ICOSL reference polypeptide isaltered in or lacks a protease cleavage site set forth as amino acids204-209 of SEQ ID NO:32. In some examples, the ICOSL referencepolypeptide contains the sequence of amino acids set forth in SEQ ID NO:545. In some aspects, the ICOSL reference polypeptide consists of thesequence of amino acids set forth in SEQ ID NO: 545.

Provided herein is a variant ICOSL Ligand (ICOSL) polypeptide containingone or more amino acid modifications in an ICOSL reference polypeptide,wherein the ICOSL reference polypeptide consists of the sequence ofamino acids set forth in SEQ ID NO: 545. Also provided herein is avariant ICOSL Ligand (ICOSL) polypeptide containing one or more aminoacid modifications in an immunoglobulin superfamily (IgSF) domain of anICOSL reference polypeptide, wherein the ICOSL reference polypeptide isaltered in one or more amino acids corresponding to amino acids 204-209with reference to SEQ ID NO: 32. In some of any of the providedembodiments, the variant ICOSL polypeptide exhibits altered binding toone or more of its binding partner(s) compared to the binding of theICOSL reference polypeptide for the one or more binding partner(s). Insome of any of the provided embodiments, the variant ICOSL polypeptideexhibits increased binding to one or more of its binding partner(s)compared to the binding of the ICOSL reference polypeptide for the oneor more binding partner(s).

In some of any of the provided embodiments, the alteration (e.g.,modification) includes a deletion of one or more contiguous amino acidscorresponding to amino acids 204-209 with reference to SEQ ID NO: 32. Insome cases, the ICOSL reference polypeptide comprises the sequence ofamino acids set forth in any of SEQ ID NOS: 600-605. In some of any ofthe provided embodiments, the ICOSL reference polypeptide consists ofthe sequence of amino acids set forth in any of SEQ ID NOS: 600-605.

In some of any of the provided embodiments, the alteration (e.g.,modification) includes at least one amino acid substitution at one orboth of position 207 and 208 corresponding to positions set forth in SEQID NO: 32. In some examples, the at least one amino acid substitution isN207A, N207G or L208G, or a conservative amino acid substitutionthereof.

In some of any of the provided embodiments, the reference ICOSLpolypeptide contains the sequence of amino acids set forth in any of SEQID NOS: 623-628. In some of any of the provided embodiments, thereference ICOSL polypeptide consists of the sequence of amino acids setforth in any of SEQ ID NOS: 623-628.

In some of any of the provided embodiments, the variant ICOSLpolypeptide exhibits reduced proteolytic cleavage when expressed from acell. In some examples, the cell is a mammalian cell. In some cases, thecell is a Chinese Hamster Ovary (CHO) cell line or a derivative thereof.

In some of any such embodiments, the amino acid modification is an aminoacid substitution, insertion or deletion. In some of any of the providedembodiments, the one or more amino acid modifications are in a positioncorresponding to position(s) selected from 10, 11, 13, 16, 18, 20, 25,27, 30, 33, 37, 38, 42, 43, 47, 52, 54, 57, 61, 62, 67, 71, 72, 74, 75,77, 78, 80, 84, 89, 90, 92, 93, 94, 96, 97, 98, 99, 100, 102, 103, 107,109, 110, 111, 113, 115, 116, 117, 119, 120, 121, 122, 126, 129, 130,132, 133, 135, 138, 139, 140, 142, 143, 144, 146, 148, 151, 152, 153,154, 155, 156, 158, 161, 164, 166, 168, 172, 173, 175, 190, 192, 193,194, 198, 201, 203, 207, 208, 210, 212, 217, 218, 220, 221, 224, 225, or227 with reference to SEQ ID NO:32. In some of any of the providedembodiments, the one or more amino acid modifications are in a positioncorresponding to position(s) selected from 10, 11, 13, 16, 18, 20, 25,26, 27, 30, 33, 37, 38, 42, 43, 47, 52, 54, 57, 61, 62, 67, 71, 72, 74,75, 77, 78, 80, 84, 89, 90, 92, 93, 94, 96, 97, 98, 99, 100, 102, 103,107, 109, 110, 111, 113, 115, 116, 117, 119, 120, 121, 122, 126, 129,130, 132, 133, 135, 137, 138, 139, 140, 142, 143, 144, 146, 151, 152,153, 154, 155, 156, 158, 161, 164, 166, 168, 172, 173, 175, 190, 192,193, 194, 198, 201, 203, 207, 208, 210, 212, 217, 218, 220, 221, 224,225, or 227 with reference to SEQ ID NO:32.

In some of any of the provided embodiments, the one or more amino acidmodifications are selected from M10V, M10I, V11E, S13G, E16V, S18R,A20V, S25G, F27S, F27C, N30D, Y33del, Q37R, K42E, T43A, Y47H, N52A,N52C, N52D, N52G, N52H, N52K, N52L, N52M, N52Q, N52R, N52S, N52T, N52V,N52Y, S54A, S54P, N57A, N57D, N57E, N57F, N57H, N57K, N57L, N57M, N57P,N57Q, N57S, N57T, N57V, N57Y, N57W, R61S, R61C, Y62F, L67P, A71T, G72R,L74Q, R75Q, D77G, F78L, L80P, N84Q, E90A, K92R, F93L, H94E, H94D, L96F,L96I, V97A, L98F, S99G, Q100A, Q100D, Q100G, Q100K, Q100L, Q100M, Q100N,Q100P, Q100R, Q100S, Q100T, Q100V, L102R, G103E, V107A, V107I, S109G,S109N, V110D, V110N, V110A, E111del, T113E, H115R, H115Q, V116A, A117T,N119Q, F120I, S121G, V122A, V122M, F120S, S126T, S126R, H129P, S130G,S132F, Q133H, E135K, F138L, T139S, C140del, C140D, S142F, I143V, I143T,N144D, Y146C, V151A, Y152C, Y152H, W153R, I154F, N155H, N155Q, K156M,D158G, L161P, L161M, L166Q, N168Q, F172S, L173S, M175T, T190A, T190S,S192G, V193M, N194D, C198R, N201S, L203P, L203F, N207Q, L208P, V210A,S212G, D217V, I218T, I218N, E220G, R221G, R221I, 1224V, T225A, N227K, ora conservative amino acid substitution thereof.

In some of any of the provided embodiments, the one or more amino acidmodifications are selected from M10V, M10I, V11E, S13G, E16V, S18R,A20T, A20V, S25G, R26S, F27C, F27S, N30D, Y33del, Q37R, T38P, K42E,T43A, Y47H, N52A, N52C, N52D, N52G, N52H, N52K, N52L, N52M, N52P, N52Q,N52R, N52S, N52T, N52V, N52Y, S54A, S54F, S54P, N57A, N57D, N57E, N57F,N57H, N57K, N57L, N57M, N57P, N57Q, N57S, N57T, N57V, N57W, N57Y, R61C,R61S, Y62F, L67P, A71T, G72R, L74Q, R75Q, D77G, F78L, L80P, N84Q, D89G,E90A, K92R, F93L, H94D, H94E, L96F, L96I, V97A, L98F, S99G, Q100A,Q100D, Q100E, Q100G, Q100K, Q100L, Q100M, Q100N, Q100P, Q100R, Q100S,Q100T, Q100V, L102R, G103E, V107A, V107I, S109G, S109N, V110A, V110D,V110N, E111del, T113E, H115Q, H115R, V116A, A117T, N119Q, F120I, F120S,S121G, V122A, V122M, S126R, S126T, H129P, S130G, S132F, Q133H, E135K,T137A, F138L, T139S, C140del, C140D, S142F, I143T, I143V, N144D, Y146C,V151A, Y152C, Y152H, W153R, I154F, N155H, N155Q, K156M, D158G, L161M,L161P, Q164L, L166Q, N168Q, F172S, L173S, M175T, T190A, T190S, S192G,V193A, V193M, N194D, C198R, N201S, L203F, L203P, N207Q, L208P, V210A,S212G, D217G, D217V, I218N, I218T, E220G, R221G, R221I, R221K, I224V,T225A, T225S, N227K, or a conservative amino acid substitution thereof.

In some of any of the provided embodiments, the one or more amino acidmodifications are in a position corresponding to position(s) 52, 57 or100. In some of any of the provided embodiments, the one or more aminoacid modifications are selected from N52A, N52C, N52D, N52G, N52H, N52K,N52L, N52M, N52Q, N52R, N52S, N52T, N52V, N52Y, N57A, N57D, N57E, N57F,N57H, N57K, N57L, N57M, N57P, N57Q, N57S, N57T, N57V, N57Y, N57W, Q100A,Q100D, Q100G, Q100K, Q100L, Q100M, Q100N, Q100P, Q100R, Q100S, Q100T orQ100V. In some of any of the provided embodiments, the one or more aminoacid modifications are selected from N52A, N52C, N52D, N52G, N52H, N52K,N52L, N52M, N52Q, N52R, N52S, N52T, N52V, N52Y, S54A, S54P, N57A, N57D,N57E, N57F, N57H, N57K, N57L, N57M, N57P, N57Q, N57S, N57T, N57V, N57Y,N57W, Q100A, Q100D, Q100G, Q100K, Q100L, Q100M, Q100N, Q100P, Q100R,Q100S, Q100T or Q100V. In some examples, the one or more amino acidmodifications are selected from among N52Y/N57Y/F138L/L203P,N52H/N57Y/Q100P, N52S/Y146C/Y152C, N52H/C198R, N52H/C140D/T225A,N52H/C198R/T225A, N52H/K92R, N52H/S99G, N57Y/Q100P, N52S/S130G/Y152C,N52S/Y152C, N52S/C198R, N52Y/N57Y/Y152C, N52Y/N57Y/H129P/C198R,N52H/L161P/C198R, N52S/T113E, N52D/S54P, N52K/L208P, N52S/Y152H,N52D/V151A, N52H/I143T, N52S/L80P, N52S/R75Q/L203P, N52S/D158G,N52D/Q133H, N52S/N57Y/H94D/L96F/L98F/Q100R,N52S/N57Y/H94D/L96F/L98F/Q100R/G103E/F120S, N52H/F78L/Q100R,N52H/N57Y/Q100R/V110D, N52H/N57Y/R75Q/Q100R/V110D, N52H/N57Y/Q100R,N52H/N57Y/L74Q/Q100R/V110D, N52H/Q100R, N52H/S121G,A20V/N52H/N57Y/Q100R/S109G, N52H/N57Y/R61S/Q100R/V110D/L173S,N52H/N57Y/Q100R/V122A, N52H/N57Y/Q100R/F172S, N52H/N57Y, N52S/F120S,N52S/V97A, N52S/G72R, N52S/A71T/A117T, N52S/E220G,Y47H/N52S/V107A/F120S, N52H/N57Y/Q100R/V110D/S132F/M175T,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R,Q37R/N52H/N57Y/Q100R/V110N/S142F/C198R/D217V/R221G,N52H/N57Y/Q100R/V110D/C198R,N52H/N57Y/Q100R/V110D/V116A/L161M/F172S/S192G/C198R,F27S/N52H/N57Y/V110N, N52S/H94E/L96I/S109N/L166Q,S18R/N52S/F93L/I143V/R221G, A20T/N52D/Y146C/Q164L,V11E/N30D/N52H/N57Y/H94E/L96I/L98F/N194D/V210A/I218T,N52S/H94E/L96I/V122M, N52H/N57Y/H94E/L96I/F120I/S126T/W153R/I218N,M10V/S18R/N30D/N52S/S126R/T139S/L203F, S25G/N30D/N52S/F120S/N227K,N30D/N52S/L67P/Q100K/D217G/R221K/T225S,N52H/N57Y/Q100R/V110D/A117T/T190S/C198R,N52H/N57Y/Q100R/V110D/F172S/C198R,S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173S/C198R,N52H/N57Y/V110A/C198R/R221I,M10I/S13G/N52H/N57Y/D77G/V110A/H129P/I143V/F172S/V193M/C198R,N52H/N57Y/R61C/Y62F/Q100R/V110N/F120S/C198R,N52H/N57Y/Q100R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/N144D/F172S/C198R, N52S/H94E/L98F/Q100R,N52S/E90A, N30D/K42E/N52S, N52S/F120S/I143V/I224V,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/S54P, T38P/N52S/N57D,N52H/C140del/T225A, N52H/F78L/Q100R/C198R, N52H/N57Y/R75Q/Q100P/V110D,N52H/N57Y/L74Q/V110D/S192G, N52H/S121G/C198R, N52S/F120S/N227K,N52S/A71T/A117T/T190A/C198R, T43A/N52H/N57Y/L74Q/D89G/V110D/F172S,N52H/N57Y/Q100R/V110D/S132F/M175T, N52D,N52H/N57Y/Q100R/V107I/V110D/I154F/C198R/R221G, N52Q/N207Q, N52Q/N168Q,N52Q/N84Q, N52Q/N119Q, N52Q/N84Q/N168Q, N52Q/N84Q/N207Q,N52Q/N119Q/N155Q, N52H/N84Q/N119Q, N52H/N84Q, N52H/N84Q/N168Q/N207Q,N52Q/N84Q/N155Q/N168Q, N52Q/N84Q/N119Q/N168Q, N52Q/N84Q/N119Q/N207Q,N52Q/N84Q/N119Q/N155Q, N52Q/N84Q/N119Q/N155Q/N207Q, N52Y/F138L/L203P,N57Y/Q100R/C198R, N57Y/F138L/L203P, Q100R/F138L,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R,N52H/N57Y/Q100R/L102R/H115R/F172S/C198R, N52H/V122A/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/N194D, N52H/N57Y/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/H115R, N52H/N57Y/Q100R/H115R,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/F172S, N52H/Q100R/H115R/I143T/F172S,N52H/N57Y/Q100P/H115R/F172S, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/E90A/H115R, N30D/K42E N52S/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N52S/H115R/F120S/I143V/C198R, N52S/H115R/F172S/C198R,N52H/N57Y/Q100P/C198R, N52H/N57Y/Q100P H115R/F172S/C198R,N52H/N57Y/Q100P/F172S/C198R, N52H/N57Y/Q100P/H115R,N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R, N52H/Q100R/H115R/F172S,N52H/Q100R/F172S/C198R, N52H/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/F172S/C198R, N52A/N57F/Q100S, N52A/N57H/Q100S,N52A/N57Y/Q100A, N52D/N57A/Q100A, N52D/Q100S, N52G/Q100A, N52H/Q100A,N52M/N57H/Q100S, N52M/N57W/Q100P, N52Q/N57F, N52Q/N57S/Q100A,N52R/N57L/Q100A, N52R/N57Y/Q100P, N52R/N57Y/Q100S, N52S/N57A/Q100A,N52S/N57H/Q100E, N52S/N57L/Q100S, N52S/N57M/Q100S, N52S/N57Y/Q100S,N52S/N57Y/Q100M, N52S/N57Y/Q100V, N52T/N57H/Q100S, N52T/N57H/Q100A,N52T/N57Y/Q100A, N52V/N57L/Q100A, N52H/N57Y/Q100K, N52K/N57Y/Q100R,N52L/N57H/Q100R, N52R/N57F/Q100N, N52R/N57F/Q100P, N52R/N57F/Q100R,N52R/N57F/Q100T, N52R/N57H/Q100K, N52R/N57L/Q100S, N52R/N57W/Q100K,N52R/N57W, N52R/N57Y/Q100R, N52C/N57E/Q100S, N52G/N57P/Q100D,N52G/N57V/Q100G, N52G/N57V, N52L/N57V, N52P/N57P, N52P/N57S/Q100G,N52S/N57L/Q100G, N52T/N57K/Q100P, N52V/N57T/Q100L or N57Q/Q100P.

In some of any of the provided embodiments, the one or more amino acidmodifications are selected from among N52Y/N57Y/F138L/L203P,N52H/N57Y/Q100P, N52S/Y146C/Y152C, N52H/C198R, N52H/C140D/T225A,N52H/C198R/T225A, N52H/K92R, N52H/S99G, N57Y/Q100P, N52S/S130G/Y152C,N52S/Y152C, N52S/C198R, N52Y/N57Y/Y152C, N52Y/N57Y/H129P/C198R,N52H/L161P/C198R, N52S/T113E, N52D/S54P, N52K/L208P, N52S/Y152H,N52D/V151A, N52H/I143T, N52S/L80P, F120S/Y152H/N201S, N52S/R75Q/L203P,N52S/D158G, N52D/Q133H, N52S/N57Y/H94D/L96F/L98F/Q100R,N52S/N57Y/H94D/L96F/L98F/Q100R/G103E/F120S, N52S/G103E, N52H/F78L/Q100R,N52H/N57Y/Q100R/V110D, N52H/N57Y/R75Q/Q100R/V110D, N52H/N57Y/Q100R,N52H/N57Y/L74Q/Q100R/V110D, N52H/Q100R, N52H/S121G,A20V/N52H/N57Y/Q100R/S109G, N52H/N57Y/R61S/Q100R/V110D/L173S,N52H/N57Y/Q100R/V122A, N52H/N57Y/Q100R/F172S, N52H/N57Y, N52S/F120S,N52S/V97A, N52S/G72R, N52S/A71T/A117T, N52S/E220G,Y47H/N52S/V107A/F120S, N52H/N57Y/Q100R/V110D/S132F/M175T,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R,Q37R/N52H/N57Y/Q100R/V110N/S142F/C198R/D217V/R221G,N52H/N57Y/Q100R/V110D/C198R,N52H/N57Y/Q100R/V110D/V116A/L161M/F172S/S192G/C198R,F27S/N52H/N57Y/V110N, N52S/H94E/L96I/S109N/L166Q,S18R/N52S/F93L/I143V/R221G, A20T/N52D/Y146C/Q164L,V11E/N30D/N52H/N57Y/H94E/L96I/L98F/N194D/V210A/I218T,N52S/H94E/L96I/V122M, N52H/N57Y/H94E/L961/F120I/S126T/W153R/I218N,M10V/S18R/N30D/N52S/S126R/T139S/L203F, S25G/N30D/N52S/F120S/N227K,N30D/N52S/L67P/Q100K/D217G/R221K/T225S,N52H/N57Y/Q100R/V110D/A117T/T190S/C198R,N52H/N57Y/Q100R/V110D/F172S/C198R,S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173S/C198R,N52H/N57Y/V110A/C198R/R221I,M10I/S13G/N52H/N57Y/D77G/V110A/H129P/I1143V/F172S/V193M, C198R,N52H/N57Y/R61C/Y62F/Q100R/V110N/F120S/C198R,N52H/N57Y/Q100R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/N144D/F172S/C198R, N52S/H94E/L98F/Q100R,N52S/E90A, N30D/K42E/N52S, N52S/F120S/I143V/I224V,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/S54P, T38P/N52S/N57D,N52H/C140del/T225A, N52H/F78L/Q100R/C198R, N52H/N57Y/R75Q/Q100P/V110D,N52H/N57Y/L74Q/V110D/S192G, N52H/S121G/C198R, N52S/F120S/N227K,N52S/A71T/A117T/T190A/C198R, T43A/N52H/N57Y/L74Q/D89G/V110D/F172S,N52H/N57Y/Q100R/V110D/S132F/M175T,N52H/N57Y/Q100R/V107I/V110D/I154F/C198R/R221G, N52Q/N207Q, N52Q/N168Q,N52Q/N84Q, N52Q/N9Q, N52Q/N4Q/N68Q, N52Q/N119Q, N52Q/N84Q/N168Q,N52Q/N84Q/N207Q, N52Q/N119Q/N155Q, N52H/N84Q/N119Q, N52H/N84Q,N52H/N84Q/N168Q/N207Q, N52Q/N84Q/N155Q/N168Q, N52Q/N84Q/N119Q/N168Q,N52Q/N84Q/N119Q/N207Q, N52Q/N84Q/N119Q/N155Q,N52Q/N84Q/N119Q/N155Q/N207Q, N52Y/F138L/L203P, N57Y/Q100R/C198R,N57Y/F138L/L203P, Q100R/F138L, N52H/N57Y/Q100R/H115R/C198R,N52H/N57Y/Q100R/F172S/C198R, N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R,N52H/N57Y/Q100R/L102R/H115R/F172S/C198R, N52H/V122A/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/N194D, N52H/N57Y/H115R/F172S/C198R,N52H/N57Y/H115R, N52H/N57Y/Q100R/H115R,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/F172S, N52H/Q100R/H115R/I143T/F172S,N52H/N57Y/Q100P/H115R/F172S, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/E90A/H115R, N30D/K42E N52S/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N52S/H115R/F120S/I143V/C198R, N52S/H115R/F172S/C198R,N52H/N57Y/Q100P/C198R, N52H/N57Y/Q100P/H115R/F172S/C198R,N52H/N57Y/Q100P/F172S/C198R, N52H/N57Y/Q100P/H115R,N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R, N52H/Q100R/H115R/F172S,N52H/Q100R/F172S/C198R, N52H/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/F172S/C198R, N52A/N57F/Q100S, N52A/N57H/Q100S,N52A/N57Y/Q100A, N52D/N57A/Q100A, N52D/Q100S, N52G/Q100A, N52H/Q100A,N52M/N57H/Q100S, N52M/N57W/Q100P, N52Q/N57F, N52Q/N57S/Q100A,N52R/N57L/Q100A, N52R/N57Y/Q100P, N52R/N57Y/Q100S, N52S/N57A/Q100A,N52S/N57H/Q100E, N52S/N57L/Q100S, N52S/N57M/Q100S, N52S/N57Y/Q100S,N52S/N57Y/Q100M, N52S/N57Y/Q100V, N52T/N57H/Q100S, N52T/N57H/Q100A,N52T/N57Y/Q100A, N52V/N57L/Q100A, N52H/N57Y/Q100K, N52K/N57Y/Q100R,N52L/N57H/Q100R, N52R/N57F/Q100N, N52R/N57F/Q100P, N52R/N57F/Q100R,N52R/N57F/Q100T, N52R/N57H/Q100K, N52R/N57L/Q100S, N52R/N57W/Q100K,N52R/N57W, N52R/N57Y/Q100R, N52C/N57E/Q100S, N52G/N57P/Q100D,N52G/N57V/Q100G, N52G/N57V, N52L/N57V, N52P/N57P, N52P/N57S/Q100G,N52S/N57L/Q100G, N52T/N57K/Q100P, N52V/N57T/Q100L, N57Q/Q100P, orR26S/N52H/N57Y/V110D/T137A/C198R.

In some of any of the provided embodiments, the one or more amino acidmodifications are N52H/Q100R. In some of any of the providedembodiments, the variant ICOSL polypeptide has the sequence set forth inSEQ ID NO:567.

In some of any of the provided embodiments, the one or more amino acidmodifications are N52H/N57Y/Q100R. In some of any of the providedembodiments, the variant ICOSL polypeptide contains the sequence setforth in SEQ ID NO:565.

In some of any of the provided embodiments, the one or more amino acidmodifications are N52L/N57H/Q100R. In some of any of the providedembodiments, the variant ICOSL polypeptide contains the sequence setforth in SEQ ID NO: 761.

In some of any of the provided embodiments, the one or more amino acidmodifications is N52D. In some of any of the provided embodiments, thevariant ICOSL polypeptide contains the sequence set forth in SEQ ID NO:548.

In some of any of the provided embodiments, the one or more amino acidmodifications is N52H/N57Y/Q100P. In some of any of the providedembodiments, the variant ICOSL polypeptide contains the sequence setforth in SEQ ID NO: 570.

In some of any of the provided embodiments, the one or more amino acidmodifications are selected from among F120S/Y152H/N201S, E111del,Y33del, N168Q/N207Q, N84Q/N207Q, N155Q/N207Q, N119Q/N168Q, N119Q/N207Q,N119Q/N155Q, N84Q/N119Q, N84Q/N155Q/N168Q, N84Q/N168Q/N207Q,N84Q/N155H/N207Q, N155Q/N168Q/N207Q, N119Q N155Q/N168Q,N119Q/N168Q/N207Q, N84Q/N119Q/N207Q, N119Q/N155H/N207Q,N84Q/N119Q/N155Q, N84Q/N119Q/N155Q/N168Q, N84Q/N155Q/N168Q/N207Q,N84Q/N119Q/N155Q/N207Q, N84Q/N119Q/N155Q/N168Q/N207Q or F138L/L203P.

In some of any such embodiments, the one or more amino acidmodifications are selected from C198R, D158G, E16V, E90A, F120S, F138L,F172S, H115R, H115X, I143T, 1143V, I224V, K156M, K42E, K92R, L102R,L203P, L208P, N194D, N30D, N52A, N52D, N52G, N52H, N52K, N52L, N52M,N52Q, N52R, N52S, N52T, N52Y, N57F, N57H, N57K, N57L, N57M, N57P, N57S,N57V, N57W, N57Y, Q100A, Q100D, Q100E, Q100K, Q100M, Q100P, Q100R,Q100S, Q100T, Q133H, R221I, R75Q, S54A, S54P, T113E, T225A, V110D,V122A, Y146C, Y152C, A117T, A20V, A71T, A91G, A91G, AE88D, C140del,C198R, D158G, D77G, D90K, E117G, E135K, E16V, E81A, E88D, E90A, F120I,F120S, F138L, F172S, F27C, F92Y, G72R, H115R, H115X, H129P, H94E, 1118V,I127T, I143T, I143V, I154F, I218N, I218T, I224V, K156M, K169E, K36G,K42E, K89R, K92R, K93R, L102R, L161P, L166Q, L173S, L203F, L203P, L208P,L209P, L40M, L70Q, L70R, L74Q, L80P, L96I, L98F, M10I, M10V, N115Q,N119Q, N122S, N144D, N155X, N168Q, N168X, N178S, N194D, N207Q, N207X,N227K, N25S, N30D, N52V, N57A, N57F, N57H, N57L, N57M, N57S, N57V, N57W,N57Y, N63S, N84Q, Q100G, Q100N, Q100V, R221G, S109G, S109N, S114T,S121G, S126R, S126T, S130G, S132F, S13G, S18R, S192G, S212G, S25G, S54A,S54P, S99G, T113E, T120S, T130A, T139S, T190A, T199S, T225A, T41I,V107I, V110A, V110D, V11E, V122A, V122M, V193M, V210A, W153R, Y146C,Y152C, or Y152H.

In some of any of the provided embodiments, the one or more amino acidmodifications are selected from among N52S, N52H, N52D, N52H/N57Y/Q100P,N52S/Y146C/Y152C, N52H/C198R, N52H/C198R/T225A, N52H/K92R, N57Y,N52S/C198R, N52S/T113E, S54A, N52D/S54P, N52K/L208P, N52H/I143T,N52S/R75Q/L203P, N52S/D158G, N52D/Q133H, N52H/N57Y/Q100R/V122A,N52H/N57Y/Q100R/F172S, N52H/N57Y/Q100R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/E90A,N52S/F120S/I143V/I224V, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/H115R/F120S/I143V/C198R, N52H/N57Y/Q100P/C198R,N52H/N57Y/Q100P/H115R/F172S/C198R, N52H/N57Y/Q100P/F172S/C198R,N52H/N57Y/Q100P/H115R, N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R,N52H/Q100R/H115X/F172S/C198R, N52H/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/H115R/F172S/C198R, Q100R, N52Y/F138L/L203P,N57Y/Q100R/C198R, N57Y/F138L/L203P, N57Y/Q100P, Q100R/F138L,N52H/N57Y/Q100R/H115R, N52H/N57Y/Q100R/F172S,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R, N52H/N57Y/Q100R/L102R,H115R/F172S/C198R, N52H/N57Y/Q100R/H115R F172S/N194D,N52H/N57Y/H115R/F172S/C198R, N52H/N57Y/Q100R/H115R/C198R,N52H/N57Y/H115R, N52H/Q100R/H115R/I143T F172S,N52H/N57Y/Q100P/H115R/F172S, E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,N52S/E90A/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N30D/K42E/N52S/H115R, N52S/E90A/H115R, N30D/K42E/N52S/H115R,N52A/N57H/Q100S, N52A/N57Y/Q100A, N52D/Q100S, N52G/Q100A,N52M/N57H/Q100S, N52M/N57W/Q100P, N52Q/N57S/Q100A, N52R/N57L/Q100A,N52S/N57H/Q100E, N52S/N57L/Q100S, N52S/N57M/Q100S, N52S/N57Y/Q100M,N52T/N57H/Q100S, N52R/N57F/Q100P, N52R/N57F/Q100T, N52R/N57W/Q100K,N52R/N57W, N52G/N57P/Q100D, N52G/N57V/Q100G, N52G/N57V, N52L/N57V,N52S/N57L/Q100G or N52T/N57K/Q100P; or N52S, N52H, N52D,N52Y/N57Y/F138L/L203P, N52H/N57Y/Q100P, N52S/Y146C/Y152C, N52H/C198R,N52H/C140del/T225A, N52H/C198R/T225A, N52H/K92R, N52H/S99G, N57Y,N57Y/Q100P, N52S/S130G/Y152C, N52S/Y152C, N52S/C198R, N52Y/N57Y/Y152C,N52Y/N57Y/H129P/C198R, N52H/L161P/C198R, N52S/T113E, S54A, N52D/S54P,N52K/L208P, N52S/Y152H, N52H/I143T, N52S/L80P, N52S/D158G, N52D/Q133H,L70Q/A91G/N144D, L70Q/A91G/E117G/I118V/T120S/T130A,L70R/A91G/I118V/T120S/T130A/T199S,L70Q/E81A/A91G/I118V/T120S/I127T/T130A,N63S/L70Q/A91G/S114T/I118V/T120S/T130A, T41I/A91G,E88D/K89R/D90K/A91G/F92Y/K93R/N122S/N178S,E88D/K89R/D90K/A91G/F92Y/K93R, AE88D/K89R/D90K/A91G/F92Y/K93R,K36G/L40M, N52H/N57Y/Q100R/V122A, N52H/N57Y/Q100R/F172S,N52H/N57Y/Q100R, N52S/F120S/N227K, N52S/N194D, N52S/F120S, N52S/G72R,N52S/A71T/A117T/T190A/C198R,N52H/N57Y/Q100R/V107I/V110D/S132F/I154F/C198R/R221G,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R,N52H/N57Y/Q100R/V110D/C198R,V11E/N30D/N52H/N57Y/H94E/L96I/L98F/N194D/V210A/I218T,N52S/H94E/L96I/V122M, N52H/N57Y/H94E/L96I/F120I/S126T/W153R/I218N,M10V/S18R/N30D/N52S/S126R/T139S/L203F, S25G/N30D/N52S/F120S/N227K,N52H/N57Y/Q100R/V110D/F172S/C198R,S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173S/C198R,N52H/N57Y/V110A/C198R/R221I,M10I/S13G/N52H/N57Y/D77G/V110A/H129P/I143V/F172S/V193M, C198R,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/N144D/F172S/C198R, N52S/H94E/L98F/Q100R,N52S/E90A, N52S/F120S/I143V/I224V, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/H115R/F120S/I143V/C198R, N52H/N57Y/Q100P/C198R,N52H/N57Y/Q100P/H115R/F172S/C198R, N52H/N57Y/Q100P/F172S/C198R,N52H/N57Y/Q100P/H115R, N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R,N52H/Q100R/H115R/F172S, N52H/Q100R/H115X/F172S/C198R,N52H/Q100R/H115R/F172S/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/H115R/F172S/C198R, N52S/H94E/L96I/S109N/L166Q/,N52H/N57Y/Q100R/C198R, N52H/N57Y/L74Q/V110D/S192G, N52H/Q100R,N52H/S121G/C198R, A20V/N52H/N57Y/Q100R/S109G, N52H/N57Y/Q100P/C198R,N52H/N57Y/Q100R/V110D/C198R/S212G, L70Q/A91G/I118A/T120S/T130A/K169E,Q100R, N52Y/F138L/L203P, N57Y/Q100R/C198R, N57Y/F138L/L203P, N52H, N57Y,N57Y/Q100P, N52H/N57Y/Q100R/H115R, N52H/N57Y/Q100R/F172S,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R, N52H/N57Y/Q100R/L102RH115R/F172S/C198R, N52H/N57Y/Q100R/H115R F172S/N194D,N52H/N57Y/H115R/F172S/C198R, N52H/N57Y/Q100R/H115R/C198R,N52H/N57Y/H115R, N52H/Q100R/H115R/I143T F172S,N52H/N57Y/Q100P/H115R/F172S, E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,N30D/K42E/N52S/H115R/C198R R221I, N52S/E90A/H115R, N30D/K42E/N52S/H115R,N52S/H115R/F172S/C198R, N119Q, N207Q, N52Q/N207X, N168X/N207X,N52Q/N168Q, N84Q/N207Q, N119Q N155X, N52Q/N119Q, N52Q/N84Q/N207Q,N119Q/N155Q/N168Q, N52H/N84Q/N119Q, N52Q/N84Q/N155X/N168X,N52A/N57F/Q100S, N52A/N57H/Q100S, N52A/N57Y/Q100A, N52D/N57A/Q100A,N52D/Q100S, N52G/Q100A, N52H/Q100A, N52M/N57H/Q100S, N52M/N57W/Q100P,N52Q/N57F, N52Q/N57S/Q100A, N52R/N57L/Q100A, N52R/N57Y/Q100P,N52R/N57Y/Q100S, N52S/N57A/Q100A, N52S/N57H/Q100E, N52S/N57L/Q100S,N52S/N57M/Q100S, N52S/N57Y/Q100S, N52S/N57Y/Q100M, N52S/N57Y/Q100V,N52T/N57H/Q100S, N52T/N57H/Q100A, N52T/N57Y/Q100A, N52V/N57L/Q100A,N52H/N57Y/Q100K, N52K/N57Y/Q100R, N52L/N57H/Q100R, N52R/N57F/Q100N,N52R/N57F/Q100P, N52R/N57F/Q100R, N52R/N57F/Q100T, N52R/N57L/Q100S,N52R/N57W/Q100K, N52R/N57W, N52G/N57V, N52L/N57V, N52S/N57L/Q100G, orN52T/N57K/Q100P. In some of any of the provided embodiments, the variantICOSL polypeptide exhibits increased binding to the ectodomain of ICOSor CD28 compared to the binding of the reference ICOSL polypeptide tothe same ectodomain.

In some of any of the provided embodiments, the one or more amino acidmodifications are selected from C198R, D158G, E16V, E90A, F120S, F138L,F172S, H115R, I143V, I224V, K156M, K42E, K92R, L102R, L203P, L208P,N194D, N30D, N52A, N52D, N52G, N52H, N52K, N52L, N52M, N52Q, N52R, N52S,N52T, N52Y, N57F, N57H, N57L, N57M, N57S, N57V, N57W, N57Y, Q100A,Q100E, Q100G, Q100K, Q100M, Q100P, Q100R, Q100S, Q133H, S212G, S54A,S54P, T113E, V110D, V122A, Y146C, Y152C, or T225A.

In some examples, the one or more amino acid modifications are selectedfrom among N52A/N57Y/Q100A, N52D/Q100S, N52G/Q100A, N52M/N57H/Q100S,N52M/N57W/Q100P, N52Q/N57S/Q100A, N52R/N57L/Q100A, N52S/N57H/Q100E,N52S/N57L/Q100S, N52S/N57M/Q100S, N52S/N57Y/Q100M, N52T/N57H/Q100S,N52R/N57F/Q100P, N52R/N57F/Q100T, N52R/N57W/Q100K, N52R/N57W, N52G/N57V,N52L/N57V, N52S/N57L/Q100G, N52T/N57K/Q100P, N52S, N52H, N52D,N52Y/N57Y/F138L/L203P, N52H/N57Y/Q100P, N52S/Y146C/Y152C, N52H/C198R,N52H/C198R/T225A, N52H/K92R, N57Y, N52S/C198R, N52S/T113E, S54A,N52D/S54P, N52K/L208P, N52H/I143T, N52S/D158G, N52D/Q133H,N52H/N57Y/Q100R/V1100D/C198R/S212G, N52H/N57Y/Q100R/V122A,N52H/N57Y/Q100R/F172S, N52H/N57Y/Q100R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/E90A,N52S/F120S/I143V/I224V, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/H115R/F120S/I143V/C198R, N52H/N57Y/Q100P/C198R,N52H/N57Y/Q100P/H115R/F172S/C198R, N52H/N57Y/Q100P/F172S/C198R,N52H/N57Y/Q100P/H115R, N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R,N52H/Q100R/H115R/F172S, N52H/Q100R/H115X/F172S/C198R,N52H/Q100R/H115R/F172S/C198R, N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S, N52H/N57Y/Q100R/H115R/F172S/C198R, Q100R,N52Y/F138L/L203P, N57Y/Q100R/C198R, N57Y/F138L/L203P, N52H, N57Y,N57Y/Q100P, Q100R/F138L, N52H/N57Y/Q100R/H115R, N52H/N57Y/Q100R/F172S,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R, N52H/N57Y/Q100R/L102RH115R/F172S/C198R, N52H/N57Y/Q100R/H115R F172S/N194D,N52H/N57Y/H115R/F172S/C198R, N52H/N57Y/Q100R/H115R/C198R,N52H/N57Y/H115R, N52H/Q100R/H115R/I143T/F172S,N52H/N57Y/Q100P/H115R/F172S, E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,N52S/E90A/H115R, N52S/E90A/H115R, or N30D/K42E/N52S/H115R. In some ofany of the provided embodiments, the variant ICOSL polypeptide exhibitsincreased binding to the ectodomain of ICOS and CD28 compared to thebinding of the reference ICOSL polypeptide to the same ectodomains.

In some of any of the provided embodiments, the variant ICOSLpolypeptide contains the sequence of amino acids set forth in any one ofSEQ ID NOS: 546-599, 734-781, 783, 786, 788, 792, 796, 798, 800, 802,804, 806, 808, 811, 813, 815, 817, 818, 820, 822, 824, 826, 827, 829,831, 833, 834, 836, 838, 840-843, 845, 847, 848, 850-853, 855, 857, 907,910, or a sequence of amino acids that exhibits at least 90%, 91%%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQID NOS: 546-599, 734-781, 783, 786, 788, 792, 796, 798, 800, 802, 804,806, 808, 811, 813, 815, 817, 818, 820, 822, 824, 826, 827, 829, 831,833, 834, 836, 838, 840-843, 845, 847, 848, 850-853, 855, 857, 907, 910.In some of any of the provided embodiments, the variant ICOSLpolypeptide consists of the sequence of amino acids set forth in any oneof SEQ ID NOS: 546-599, 734-781, 783, 786, 788, 792, 796, 798, 800, 802,804, 806, 808, 811, 813, 815, 817, 818, 820, 822, 824, 826, 827, 829,831, 833, 834, 836, 838, 840-843, 845, 847, 848, 850-853, 855, 857, 907,910, or a sequence of amino acids that exhibits at least 90%, 91%%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQID NOS: 546-599, 734-781, 783, 786, 788, 792, 796, 798, 800, 802, 804,806, 808, 811, 813, 815, 817, 818, 820, 822, 824, 826, 827, 829, 831,833, 834, 836, 838, 840-843, 845, 847, 848, 850-853, 855, 857, 907, 910.

In some of any of the provided embodiments of the variant ICOS Ligand(ICOSL) polypeptide containing an IgV domain or specific bindingfragment thereof, an IgC domain or a specific binding fragment thereof,or both, the variant ICOSL polypeptide contains one or more amino acidmodifications in an ICOSL reference polypeptide or a specific bindingfragment thereof corresponding to amino acid modifications are selectedfrom N52A, N52C, N52D, N52G, N52K, N52L, N52M, N52R, N52T, N52V, N57A,N57E, N57F, N57H, N57K, N57L, N57M, N57P, N57Q, N57S, N57T, N57V, N57W,Q100A, Q100D, Q100G, Q100L, Q100M, Q100N, Q100R, Q100S, Q100T or Q100Vwith reference to SEQ ID NO:32. In some of any of the providedembodiments, the one or more amino acid modifications are selected fromamong N52A/N57F/Q100S, N52A, /N57H/Q100S, N52A/N57Y/Q100A,N52D/N57A/Q100A, N52D/Q100S, N52G/Q100A, N52H/Q100A, N52M/N57H/Q100S,N52M/N57W/Q100P, N52Q/N57F, N52Q/N57S/Q100A, N52R/N57L/Q100A,N52R/N57Y/Q100P, N52R/N57Y/Q100S, N52S/N57A/Q100A, N52S/N57H/Q100E,N52S/N57L/Q100S, N52S/N57M/Q100S, N52S/N57Y/Q100S, N52S/N57Y/Q100M,N52S/N57Y/Q100V, N52T/N57H/Q100S, N52T/N57H/Q100A, N52T/N57Y/Q100A,N52V/N57L/Q100A, N52H/N57Y/Q100K, N52K/N57Y/Q100R, N52L/N57H/Q100R,N52R/N57F/Q100N, N52R/N57F/Q100P, N52R/N57F/Q100R, N52R/N57F/Q100T,N52R/N57H/Q100K, N52R/N57L/Q100S, N52R/N57W/Q100K, N52R/N57W,N52R/N57Y/Q100R, N52C/N57E/Q100S, N52G/N57P/Q100D, N52G/N57V/Q100G,N52G/N57V, N52L/N57V, N52P/N57P, N52P/N57S/Q100G, N52S/N57L/Q100G,N52T/N57K/Q100P, N52V/N57T/Q100L or N57Q/Q100P.

In some of any of the provided embodiments, the ICOSL referencepolypeptide is a mammalian ICOSL or a specific binding fragment thereof.In some examples, the ICOSL reference polypeptide is a human ICOSL or aspecific binding fragment thereof.

In some of any of the provided embodiments, the ICOSL referencepolypeptide contains (i) the sequence of amino acids set forth in SEQ IDNO:32, (ii) a sequence of amino acids that has at least 95% sequenceidentity to SEQ ID NO:32; or (iii) a portion of (i) or (ii) comprisingan IgV domain or IgC domain or specific binding fragments thereof orboth. In some of any of the provided embodiments, the specific bindingfragment of the IgV domain or IgC domain has a length of at least 50,60, 70, 80, 90, 100, 110 or more amino acids; or the specific bindingfragment of the IgV domain contains a length that is at least 80% of thelength of the IgV domain set for as amino acids 19-129 of SEQ ID NO:5and/or the specific binding fragment of the IgC domain comprises alength that is at least 80% of the length of the IgC domain set forth asamino acids 141-227 of SEQ ID NO:5. In some of any of the providedembodiments, the variant ICOSL polypeptide comprises the IgV domain or aspecific fragment thereof and the IgC domain or a specific fragmentthereof.

In some of any of the provided embodiments, the variant ICOSLpolypeptide contains the sequence of amino acids set forth in any one ofSEQ ID NOS: 638-685, or a sequence of amino acids that exhibits at least90%, 91%%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identityto any one of SEQ ID NOS: 638-685. In some of any of the providedembodiments, the variant ICOSL polypeptide consists of the sequence ofamino acids set forth in any one of SEQ ID NOS: 638-685, or a sequenceof amino acids that exhibits at least 90%, 91%%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOS:638-685.

In some of any of the provided embodiments, the variant ICOSLpolypeptide contains the IgV domain or a specific binding fragmentthereof. In some of any of the provided embodiments, the variant ICOSLpolypeptide contains the sequence of amino acids set forth in any one ofSEQ ID NOS: 686-781, or a sequence of amino acids that exhibits at least90%, 91%%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identityto any one of SEQ ID NOS: 686-781. In some of any of the providedembodiments, the variant ICOSL polypeptide consists of the sequence ofamino acids set forth in any one of SEQ ID NOS: 686-781, or a sequenceof amino acids that exhibits at least 90%, 91%%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOS:686-781.

In some of any of the provided embodiments, the IgV domain or specificbinding fragment thereof is the only ICOSL portion of the variant ICOSLpolypeptide. In some examples, the IgC domain or specific bindingfragment thereof is the only ICOSL portion of the variant ICOSLpolypeptide.

In some of any of the provided embodiments, the variant ICOSLpolypeptide exhibits altered binding to the ectodomain of ICOS or CD28compared to the binding of the ICOSL reference polypeptide for the sameectodomain. In some aspects, the variant ICOSL polypeptide exhibitsincreased binding to the ectodomain(s) of ICOS or CD28 compared to thebinding of the ICOSL reference polypeptide for the same ectodomain(s).In some examples, the binding is increased more than 1.2-fold, 1.5-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold or 60-fold.

In some of any such embodiments, the ICOS is a human ICOS. In some ofany of the provided embodiments, the CD28 is a human CD28.

In some of any of the provided embodiments, the variant ICOSLpolypeptide exhibits decreased binding to the ectodomain of CTLA-4compared to the binding of the reference ICOSL polypeptide for the sameectodomain. In some examples, the binding is decreased more than1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or 60-fold.In some of any of the provided embodiments, the CTLA-4 is a humanCTLA-4.

In some of any of the provided embodiments, the altered (increased ordecreased) binding is altered (increased or decreased) binding affinity.In some of any such embodiments, the variant ICOSL polypeptide comprisesup to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19or 20 amino acid modifications, optionally amino acid substitutions,insertions and/or deletions. In some cases, the variant ICOSLpolypeptide exhibits at least or at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% sequence identity to the ICOSL referencepolypeptide.

In some of any of the provided embodiments, the variant ICOSLpolypeptide is a soluble protein. In some of any of the providedembodiments, the variant ICOSL polypeptide lacks a transmembrane domainand intracellular signaling domain; and/or when expressed from a cell,the variant ICOSL polypeptide is not expressed on the surface of thecell.

In some of any of the provided embodiments, the variant ICOSLpolypeptide further contains transmembrane domain. In some cases, thetransmembrane domain contains the sequence of amino acids set forth asresidues 257-277 of SEQ ID NO:5 or a functional variant thereof thatexhibits at least 85% sequence identity to residues 257-277 of SEQ IDNO:5. In some of any of the provided embodiments, the variant ICOSLpolypeptide further contains a cytoplasmic signaling domain linked tothe transmembrane domain. In some cases, the cytoplasmic signalingdomain contains the sequence of amino acids set forth as residues278-302 of SEQ ID NO:5 or a functional variant thereof that exhibits atleast 85% sequence identity to residues 278-302 of SEQ ID NO:5.

In some of any of the provided embodiments, the variant ICOSLpolypeptide is deglycosylated or partially deglycosylated compared tothe ICOSL reference sequence.

Provided herein is an immunomodulatory protein containing any of theprovided variant ICOSL polypeptide and a half-life extending moiety. Insome of any of the provided embodiments, the half-life extending moietycomprises a multimerization domain, albumin, an albumin-bindingpolypeptide, Pro/Ala/Ser (PAS), a C-terminal peptide (CTP) of the betasubunit of human chorionic gonadotropin, polyethylene glycol (PEG), longunstructured hydrophilic sequences of amino acids (XTEN), hydroxyethylstarch (HES), an albumin-binding small molecule, or a combinationthereof. In some cases, the half-life extending moiety is or comprisesPro/Ala/Ser (PAS) and the variant ICOSL polypeptide is PASylated. Insome of any of the provided embodiments, the half-life extending moietycontains the sequence set forth in SEQ ID NO:904.

In some of any of the provided embodiments, the half-life extendingmoiety is or contains a multimerization domain. In some instances, themultimerization domain is selected from an Fc region of animmunoglobulin, a leucine zipper, an isoleucine zipper or a zinc finger.In some of any of the provided embodiments, the variant ICOSLpolypeptide is linked, directly or indirectly via a linker, to themultimerization domain.

In some of any of the provided embodiments, the immunomodulatory proteinis a multimer containing a first variant ICOSL polypeptide linked to afirst multimerization domain and a second variant ICOSL polypeptidelinked to a second multimerization domain, wherein the first and secondmultimerization domains interact to form a multimer comprising the firstand second variant ICOSL polypeptide. In some cases, the multimer is adimer.

In some of any of the provided embodiments, the first variant ICOSLpolypeptide and the second variant ICOSL polypeptide are the same. Insome of any of the provided embodiments, the dimer is a homodimer. Insome instances, the dimer is a heterodimer.

In some of any of the provided embodiments, the multimerization domainis or contains an Fc region of an immunoglobulin. In some of any of theprovided embodiments, the Fc region is of an immunoglobulin G1 (IgG1) oran immunoglobulin G2 (IgG2) protein. In some examples, theimmunoglobulin protein is human and/or the Fc region is human. In someof any of the provided embodiments, the Fc region contains the sequenceof amino acids set forth in SEQ ID NO: 227 or a variant thereof thatexhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to SEQ ID NO:227. In some aspects, the Fc regioncontains the sequence of amino acids set forth in SEQ ID NO: 226 or avariant thereof that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% sequence identity to SEQ ID NO:226. In some of anyof the provided embodiments, the Fc region exhibits one or more effectorfunctions. In some of any of the provided embodiments, the Fc regionexhibits one or more reduced effector function compared to a wildtype Fcregion, optionally wherein the wildtype human Fc is of human IgG1.

In some of any of the provided embodiments, the one or more effectorfunction is selected from among antibody dependent cellular cytotoxicity(ADCC), complement dependent cytotoxicity, programmed cell death andcellular phagocytosis. In some of any of the provided embodiments, theFc region is a variant Fc region comprising one or more amino acidsubstitutions compared to the wildtype Fc region.

In some of any of the provided examples, the one or more amino acidsubstitutions of the variant Fc region are selected from N297G,E233P/L234V/L235A/G236del/S267K or L234A/L235E/G237A, wherein theresidue is numbered according to the EU index of Kabat. In some of anyof the provided embodiments, the variant Fc region further contains theamino acid substitution C220S, wherein the residues are numberedaccording to the EU index of Kabat. In some aspects, the Fc regioncontains the sequence of amino acid sequence set forth in any of SEQ IDNOS: 476-478 or a sequence of amino acids that exhibits at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity toany of SEQ ID NOS:476-478 and contains the amino acid substitutions. Insome of any of the provided embodiments, the Fc region contains K447del,wherein the residue is numbered according to the EU index of Kabat.

In some of any of the provided embodiments, the Fc region contains thesequence of amino acid sequence set forth in any of SEQ ID NOS: 632-634or a sequence of amino acids that exhibits at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ IDNOS: 632-634 and contains the amino acid substitutions.

In some of any of the provided embodiments, the Fc region contains thesequence of amino acid sequence set forth in SEQ ID NOS: 474 or 637, ora sequence of amino acids that exhibits at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOS:474 or 637 and contains the amino acid substitutions.

In some of any of the provided embodiments, the Fc region contains thesequence of amino acids set forth in SEQ ID NO: 478 or SEQ ID NO: 634.In some of any of the provided embodiments, the Fc region contains thesequence of amino acids set forth in SEQ ID NO: 477. In some of any ofthe provided embodiments, the Fc region contains the sequence of aminoacids set forth in SEQ ID NO: 633. In some of any of the providedembodiments, the Fc region contains the sequence of amino acids setforth in SEQ ID NO: 474. In some of any of the provided embodiments, theFc region contains the sequence of amino acids set forth in SEQ IDNO:637.

Provided herein is an immunomodulatory protein containing (a) a variantICOSL polypeptide containing one or more amino acid modifications in animmunoglobulin superfamily (IgSF) domain of an ICOSL referencepolypeptide, wherein the variant ICOSL polypeptide exhibits alteredbinding to the ectodomain(s) of ICOS or CD28 compared to the binding ofthe ICOSL reference polypeptide for the same ectodomain(s); and (b) avariant Fc region containing amino acid substitutions selected fromN297G/K447del, E233P/L234V/L235A/G236del/S267K/K447del orL234A/L235E/G237A/K447del compared to wildtype human IgG1, wherein theresidues are numbered according to the EU index of Kabat. In some cases,the immunomodulatory protein is a dimer. In some of any of the providedembodiments, the variant Fc region further contains the amino acidsubstitution C220S, wherein the residues are numbered according to theEU index of Kabat. In some examples, the Fc region contains the sequenceof amino acid sequence set forth in any of SEQ ID NOS: 632-634 or asequence of amino acids that exhibits at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ IDNOS:632-634 and contains the amino acid substitutions. In some of any ofthe provided embodiments, the Fc region contains the sequence of aminoacid sequence set forth in SEQ ID NOS: 474 or 637, or a sequence ofamino acids that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to SEQ ID NOS: 474 or 637 andcontains the amino acid substitutions.

In some of any of the provided embodiments, the variant ICOSLpolypeptide is linked, directly or indirectly via a linker, to thevariant Fc region. In some examples, the linker contains 1 to 10 aminoacids. In some of any of the provided embodiments, the linker isselected from AAA, G4S (SEQ ID NO: 636), (G₄S)₂ (SEQ ID NO: 229) orGSGGGGS linker (SEQ ID NO: 635). In some of any of the providedembodiments, the linker is (G₄S)₃ (SEQ ID NO: 228).

In some of any of the provided embodiments, the linker is AAA. In someof any of the provided embodiments, the linker is G4S (SEQ ID NO:636).In some of any of the provided embodiments, the linker is (G₄S)₂ (SEQ IDNO:229). In some of any of the provided embodiments, the linker isGSGGGGS linker (SEQ ID NO: 635).

In some of any of the provided embodiments of a fusion protein, e.g. avariant ICOSL-Fc fusion protein, the variant ICOSL polypeptide is orcomprise an IgV domain. In some of any of the provided embodiments, thevariant ICOSL polypeptide contains amino acid modifications N52H/Q100R.In some of any of the provided embodiments, the variant ICOSLpolypeptide has the sequence set forth in SEQ ID NO: 567. In some of anyof the provided embodiments, the variant ICOSL polypeptide comprisesamino acid modifications N52H/N57Y/Q100R. In some of any of the providedembodiments, the variant ICOSL polypeptide has the sequence set forth inSEQ ID NO: 565. In some of any of the provided embodiments, the variantICOSL polypeptide comprises amino acid modifications areN52L/N57H/Q100R. In some of any of the provided embodiments, the variantICOSL polypeptide has the sequence set forth in SEQ ID NO: 761. In someof any of the provided embodiments, the variant ICOSL polypeptidecomprises amino acid modifications N52H/N57Y/Q100P. In some of any ofthe provided embodiments, the variant ICOSL polypeptide has the sequenceset forth in SEQ ID NO: 570. In some of any of the provided embodiments,the variant ICOSL polypeptide comprises the amino acid modification isN52D. In some of any of the provided embodiments, the polypeptide hasthe sequence set forth in SEQ ID NO: 548.

In some of any of the provided embodiments, provided is a variantICOSL-Fc fusion protein that has the sequence of amino acids set forthin SEQ ID NO: 928. In some of any of the provided embodiments, providedis a variant ICOSL-Fc fusion protein that has a sequence of amino acidsthat has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to the sequence set forthin SEQ ID NO: 928.

In particular embodiments, the provided immunomodulatory proteins, suchas fusion proteins, e.g. variant ICOSL-Fc fusion proteins, binds CD28and ICOS. In some embodiments, the variant ICOSL-Fc fusion proteinexhibits increased binding affinity to CD28 and/or ICOS compared to ahuman wild-type ICOSL-Fc fusion protein, e.g. containing an ICOSL IgVportion set forth in SEQ ID NO:545 linked via a linker, e.g. set forthin SEQ ID NO:229, to an Fc region. In such an example, the Fc region isan inert or effectorless Fc containing the mutations L234A, L235E andL235E in a human IgG1 Fc, e.g. set forth in SEQ ID NO:637,

Provided herein is an immunomodulatory protein containing any of thevariant ICOSL polypeptides linked to a second polypeptide comprising animmunoglobulin superfamily (IgSF) domain. In some cases, the IgSF domainis affinity modified and exhibits altered binding to one or more of itscognate binding partner(s) compared to the unmodified or wild-type IgSFdomain. In some of any of the provided embodiments, the IgSF domainexhibits increased binding to one or more of its cognate bindingpartner(s) compared to the unmodified or wild-type IgSF domain.

In some of any of the provided embodiments, the variant ICOSLpolypeptide is a first ICOSL variant polypeptide and the IgSF domain ofthe second polypeptide is an IgSF domain from a second variant ICOSLpolypeptide provided herein, wherein the first and second ICOSL variantare the same or different.

In some of any of the provided embodiments, the variant ICOSLpolypeptide is capable of specifically binding to CD28 or ICOS and theIgSF domain of the second polypeptide is capable of binding to a bindingpartner other than one specifically bound by the ICOSL variantpolypeptide. In some of any of the provided embodiments, the IgSF domainis from a member of the B7 family.

In some of any of the provided embodiments, the IgSF domain is atumor-localizing moiety that binds to a ligand expressed on a tumor oris an inflammatory-localizing moiety that binds to a ligand expressed ona cell or tissue of an inflammatory environment. In some cases, theligand is B7H6. In some examples, the IgSF domain is from NKp30.

In some of any of the provided embodiments, the IgSF domain of thesecond polypeptide is or comprises an IgV domain. In some of any of theprovided embodiments, the IgSF domain of the second polypeptide is avariant NKp30 molecule containing L30V/A60V/S64P/S86G. In some of any ofthe provided embodiments, the IgSF domain of the second polypeptide hasthe sequence set forth in SEQ ID NO: 504.

In some of any of the provided embodiments, the IgSF domain is orcomprises an IgV domain. In some cases, the variant ICOSL polypeptide isor contains an IgV domain.

In some of any of the provided embodiments, the variant ICOSLpolypeptide is or comprise an IgV domain. In some of any of the providedembodiments, the variant ICOSL polypeptide contains amino acidmodifications N52H/Q100R. In some of any of the provided embodiments,the variant ICOSL polypeptide has the sequence set forth in SEQ ID NO:567. In some of any of the provided embodiments, the variant ICOSLpolypeptide comprises amino acid modifications N52H/N57Y/Q100R. In someof any of the provided embodiments, the variant ICOSL polypeptide hasthe sequence set forth in SEQ ID NO: 565. In some of any of the providedembodiments, the variant ICOSL polypeptide comprises amino acidmodifications are N52L/N57H/Q100R. In some of any of the providedembodiments, the variant ICOSL polypeptide has the sequence set forth inSEQ ID NO: 761. In some of any of the provided embodiments, the variantICOSL polypeptide comprises amino acid modifications N52H/N57Y/Q100P. Insome of any of the provided embodiments, the variant ICOSL polypeptidehas the sequence set forth in SEQ ID NO: 570. In some of any of theprovided embodiments, the variant ICOSL polypeptide comprises the aminoacid modification is N52D. In some of any of the provided embodiments,the polypeptide has the sequence set forth in SEQ ID NO:548.

In some of any of the provided embodiments, the immunomodulatory proteincomprises a multimerization domain linked to one or both of the variantICOSL polypeptide or the second polypeptide comprising the IgSF domain.In some cases, the multimerization domain is an Fc domain or a variantthereof with reduced effector function.

In some of any of the provided embodiments, the immunomodulatory proteinis dimeric. In some cases, the immunomodulatory protein is homodimeric.In some aspects, the immunomodulatory protein is heterodimeric.

Provided herein is a conjugate containing any of the provided variantICOSL polypeptides or any of the provided immunomodulatory protein and aheterologous moiety. In some cases, the variant ICOSL polypeptide islinked, directly or indirectly via a linker, to the heterologous moiety.In some of any of the provided embodiments, the targeting moiety is aprotein, a peptide, nucleic acid, small molecule or nanoparticle. Insome examples, the target moiety is a protein or a peptide. In some ofany of the provided embodiments, the conjugate is a fusion protein.

Provided is a fusion protein containing any of the provided variantICOSL polypeptides or any of the provided immunomodulatory protein and aheterologous moiety. In some cases, the moiety is a targeting moietythat specifically binds to a molecule on the surface of a cell. In someexamples, the targeting moiety specifically binds to a molecule on thesurface of an immune cell. In some of any of the provided embodiments,the immune cell is an antigen presenting cell or a lymphocyte. In somecases, the targeting moiety is a tumor-localizing moiety that binds to amolecule on the surface of a tumor.

In some of any of the provided embodiments, the targeting moiety bindsto a molecule HER1/EGFR, HER2/ERBB2, CD20, CD25 (IL-2Rα receptor), CD33,CD52, CD133, CD206, CEA, CEACAM1, CEACAM3, CEACAM5, CEACAM6, cancerantigen 125 (CA125), alpha-fetoprotein (AFP), Lewis Y, TAG72, Caprin-1,mesothelin, PDGF receptor (PDGFR; such as PDGF-R α), PD-1, PD-L1,CTLA-4, IL-2 receptor, vascular endothelial growth factor (VEGF), CD30,EpCAM, EphA2, Glypican-3, gpA33, mucins, CAIX, PSMA, folate-bindingprotein, gangliosides (such as GD2, GD3, GM1 and GM2), VEGF receptor(VEGFR), VEGFR2, VEGF-A, integrin αVβ3, integrin α5β1, ERBB3, MET,IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, AFP, BCR complex,CD3, CD18, CD44, CTLA-4, gp72, HLA-DR 10β, HLA-DR antigen, IgE, MUC-1,nuC242, PEM antigen, metalloproteinases, Ephrin receptor, Ephrinligands, HGF receptor, CXCR4, CXCR4, Bombesin receptor, SK-1antigen,Bcr-ab1, RET, MET, TRKB, TIE2, ALK, ROS, EML4-ALK, ROS1, BRAFV600E, SRC,c-KIT, mTOR, TSC1, TSC2, BTK, KIT, BRCA, CDK 4/6, JAK1, JAK2, BRAF,FLT-3, MEK1, MEK2, SMO or B7-H6 (NCR3LG1). In some aspects, thetargeting moiety binds to PD-L1.

In some of any of the provided embodiments, the targeting moiety is anantibody or antigen-binding fragment. In some of any of the providedembodiments, the antibody is selected from cetuximab, panitumumab,zalutumumab, nimotuzumab, trastuzumab, Ado-trastuzumab emtansine,Tositumomab (Bexxar®), Rituximab (Rituxan, Mabthera), Ibritumomabtiuxetan (Zevalin), Daclizumab (Zenapax), Gemtuzumab (Mylotarg),Alemtuzumab, CEA-scan Fab fragment, OC125 monoclonal antibody, ab75705,B72.3, Bevacizumab (Avastin®), Afatinib, Axitinib, Bosutinib,Cabozantinib, Ceritinib, Crizotinib, Dabrafenib, Dasatinib, Dinutuximab(Unituxin™), Erlotinib, Everolimus, Ibrutinib, Imatinib, Lapatinib,Lenvatinib, Nilotinib, Olaparib, Olaratumab (Lartruvo™), Palbociclib,Pazopanib, Pertuzumab (Perjeta®), Ramucirumab (Cyramza®), Regorafenib,Ruxolitinib, Sorafenib, Sunitinib, Temsirolimus, Trametinib, Vandetanib,Vemurafenib, Vismodegib, Basiliximab, Ipilimumab, Nivolumab,pembrolizumab, MPDL3280A, Pidilizumab (CT-011), AMP-224, MSB001078C, orMEDI4736, BMS-935559, LY3300054, atezolizumab, avelumab or durvalumab oris an antigen-binding fragment thereof.

In some of any of the provided embodiments, the variant ICOSLpolypeptide is linked, directly or indirectly via a linker, to theN-terminus of the heavy and/or light chain of the antibody orantigen-binding fragment. In some cases, the variant ICOSL polypeptideis linked, directly or indirectly via a linker, to the C-terminus of theheavy and/or light chain of the antibody or antigen binding fragment.

In some of any of the provided embodiments, the conjugate is divalent,tetravalent, hexavalent or octavalent. In some of any of the providedembodiments, the heterologous moiety is or contains a label fordetection or purification of the variant ICOSL polypeptide.

Provided herein is a monovalent fusion protein containing a variantICOSL polypeptide containing one or more amino acid modifications in animmunoglobulin superfamily (IgSF) domain of an ICOSL referencepolypeptide, wherein the variant ICOSL polypeptide exhibits alteredbinding to the ectodomain(s) of ICOS or CD28 compared to the binding ofthe ICOSL reference polypeptide for the same ectodomain(s); and a labelfor detection or purification of the variant ICOSL polypeptide. In someof any of the provided embodiments, the label for detection orpurification is selected from a poly-histidine (His) tag, a FLAG-tag, aMyc-tag, or a fluorescent protein-tag.

In some of any of the provided embodiments, the variant ICOSLpolypeptide contains one or more amino acid modifications in a positioncorresponding to position(s) selected from 10, 11, 13, 16, 18, 20, 25,27, 30, 33, 37, 38, 42, 43, 47, 52, 54, 57, 61, 62, 67, 71, 72, 74, 75,77, 78, 80, 84, 89, 90, 92, 93, 94, 96, 97, 98, 99, 100, 102, 103, 107,109, 110, 111, 113, 115, 116, 117, 119, 120, 121, 122, 126, 129, 130,132, 133, 135, 138, 139, 140, 142, 143, 144, 146, 148, 151, 152, 153,154, 155, 156, 158, 161, 164, 166, 168, 172, 173, 175, 190, 192, 193,194, 198, 201, 203, 207, 208, 210, 212, 217, 218, 220, 221, 224, 225, or227 with reference to SEQ ID NO:32. In some of any of the providedembodiments, the variant ICOSL polypeptide contains one or more aminoacid modifications are in a position corresponding to position(s)selected from 10, 11, 13, 16, 18, 20, 25, 26, 27, 30, 33, 37, 38, 42,43, 47, 52, 54, 57, 61, 62, 67, 71, 72, 74, 75, 77, 78, 80, 84, 89, 90,92, 93, 94, 96, 97, 98, 99, 100, 102, 103, 107, 109, 110, 111, 113, 115,116, 117, 119, 120, 121, 122, 126, 129, 130, 132, 133, 135, 137, 138,139, 140, 142, 143, 144, 146, 151, 152, 153, 154, 155, 156, 158, 161,164, 166, 168, 172, 173, 175, 190, 192, 193, 194, 198, 201, 203, 207,208, 210, 212, 217, 218, 220, 221, 224, 225, or 227 with reference toSEQ ID NO:32.

In some cases, the one or more amino acid modifications are selectedfrom M10V, M10I, V11E, S13G, E16V, S18R, A20V, S25G, F27S, F27C, N30D,Y33del, Q37R, K42E, T43A, Y47H, N52A, N52C, N52D, N52G, N52H, N52K,N52L, N52M, N52Q, N52R, N52S, N52T, N52V, N52Y, N52K, S54A, S54P, N57A,N57D, N57E, N57F, N57H, N57K, N57L, N57M, N57P, N57Q, N57S, N57T, N57V,N57Y, N57W, R61S, R61C, Y62F, L67P, A71T, G72R, L74Q, R75Q, D77G, F78L,L80P, N84Q, E90A, K92R, F93L, H94E, H94D, L96F, L96I, V97A, L98F, S99G,Q100A, Q100D, Q100G, Q100K, Q100L, Q100M, Q100N, Q100P, Q100R, Q100S,Q100T, Q100V, L102R, G103E, V107A, V107I, S109G, S109N, V110D, V110N,V110A, E111del, T113E, H115R, H115Q, V116A, A117T, N119Q, F120I, S121G,V122A, V122M, F120S, S126T, S126R, H129P, S130G, S132F, Q133H, E135K,F138L, T139S, C140del, C140D, S142F, I143V, I143T, N144D, Y146C, V151A,Y152C, Y152H, W153R, I154F, N155H, N155Q, K156M, D158G, L161P, L161M,L166Q, N168Q, F172S, L173S, M175T, T190A, T190S, S192G, V193M, N194D,C198R, N201S, L203P, L203F, N207Q, L208P, V210A, S212G, D217V, I218T,I218N, E220G, R221G, R221I, I224V, T225A, N227K, or a conservative aminoacid substitution thereof. In some cases, the one or more amino acidmodifications are selected from M10V, M10I, V11E, S13G, E16V, S18R,A20T, A20V, S25G, R26S, F27C, F27S, N30D, Y33del, Q37R, T38P, K42E,T43A, Y47H, N52A, N52C, N52D, N52G, N52H, N52K, N52L, N52M, N52P, N52Q,N52R, N52S, N52T, N52V, N52Y, S54A, S54F, S54P, N57A, N57D, N57E, N57F,N57H, N57K, N57L, N57M, N57P, N57Q, N57S, N57T, N57V, N57W, N57Y, R61C,R61S, Y62F, L67P, A71T, G72R, L74Q, R75Q, D77G, F78L, L80P, N84Q, D89G,E90A, K92R, F93L, H94D, H94E, L96F, L96I, V97A, L98F, S99G, Q100A,Q100D, Q100E, Q100G, Q100K, Q100L, Q100M, Q100N, Q100P, Q100R, Q100S,Q100T, Q100V, L102R, G103E, V107A, V107I, S109G, S109N, V110A, V110D,V110N, E111del, T113E, H115Q, H115R, V116A, A117T, N119Q, F120I, F120S,S121G, V122A, V122M, S126R, S126T, H129P, S130G, S132F, Q133H, E135K,T137A, F138L, T139S, C140del, C140D, S142F, I143T, I143V, N144D, Y146C,V151A, Y152C, Y152H, W153R, I154F, N155H, N155Q, K156M, D158G, L161M,L161P, Q164L, L166Q, N168Q, F172S, L173S, M175T, T190A, T190S, S192G,V193A, V193M, N194D, C198R, N201S, L203F, L203P, N207Q, L208P, V210A,S212G, D217G, D217V, I218N, I218T, E220G, R221G, R221I, R221K, I224V,T225A, T225S, N227K, or a conservative amino acid substitution thereof.

In some of any of the provided embodiments, the ICOSL referencepolypeptide contains (i) the sequence of amino acids set forth in SEQ IDNO:32, (ii) a sequence of amino acids that has at least 95% sequenceidentity to SEQ ID NO:32; or (iii) a portion of (i) or (ii) comprisingan IgV domain or IgC domain or specific binding fragments thereof orboth.

In some of any of the provided embodiments, the ICOSL referencepolypeptide comprises the sequence of amino acids set forth in any ofSEQ ID NOS: 196, 545, 600-605 and 623-628. In some aspects, the ICOSLreference polypeptide consists of the sequence of amino acids set forthin any of SEQ ID NOS: 32, 196, 545, 600-605 and 623-628.

Provided is a nucleic acid molecule(s) encoding any of the providedvariant ICOSL polypeptides, immunomodulatory proteins, or fusionproteins. In some cases, the nucleic acid molecules(s) is syntheticnucleic acid. In some examples, the nucleic acid molecule(s) is cDNA.

Provided is a vector containing any of the provided the nucleic acidmolecule(s). In some cases, the vector is an expression vector. In someof any of the provided embodiments, the vector is a mammalian expressionvector or a viral vector.

Provided is a cell containing any of the provided vectors. In somecases, the cell is a mammalian cell. In some of any of the providedembodiments, the cell is a Chinese Hamster Ovary (CHO) cell or aderivative thereof.

Provided is a method of producing an immunomodulatory protein containingany of the variant ICOSL polypeptides, including introducing any of theprovided nucleic acid molecules or vectors into a host cell underconditions to express the protein in the cell. In some examples, thehost cell is a mammalian cell. In some instances, the mammalian cell isa Chinese Hamster Ovary cell or a derivative thereof. In some of any ofthe provided embodiments, the method further includes isolating orpurifying the protein from the cell.

Provided is a protein produced by any of the provided methods.

Provided is a composition containing a protein containing any of theprovided variant ICOSL polypeptides, or immunomodulatory proteins,wherein at least 95%, 96%, 97%, 98%, 99% of the individual sequences ofthe protein or the immunomodulatory protein in the composition have anidentical sequence length, optionally wherein the composition is apharmaceutical composition comprising a pharmaceutically acceptablecarrier. In some of any of the provided embodiments, the protein orimmunomodulatory protein is purified from Chinese Hamster Ovary Cells ora derivative thereof.

Provided is a polynucleotide containing a nucleic acid encoding avariant ICOSL polypeptide containing a provided transmembrane domain andone or more nucleic acid encoding one or more chain of a recombinantantigen receptor. In some cases, the recombinant antigen receptor is achimeric antigen receptor (CAR) or an engineered T cell receptor (TCR).In some of any of the provided embodiments, each of the nucleic acidencoding the variant ICOSL polypeptide and the one or more nucleic acidencoding one or more chain of the recombinant receptor is separated by anucleic acid encoding a self-cleaving peptide or a peptide that causesribosome skipping.

In some examples, the polynucleotide contains the nucleic acid encodingthe variant ICOSL polypeptide, a nucleic acid encoding a self-cleavingpeptide or a peptide that causes ribosome skipping and a nucleic acidencoding a CAR. In some examples, the polynucleotide comprises thenucleic acid encoding the variant ICOSL polypeptide, a nucleic acidencoding a first self-cleaving peptide or a peptide that causes ribosomeskipping, a nucleic acid encoding one of an engineered TCRalpha chain oran engineered TCRbeta chain, a nucleic acid encoding a secondself-cleaving peptide or a peptide that causes ribosome skipping, and anucleic acid encoding the other of the engineered TCRalpha chain or theengineered TCRbeta chain. In some aspects, the encoded first and secondself-cleaving peptide is the same. In some of any of the providedembodiments, the self-cleaving peptide or the peptide that causesribosome skipping is a T2A, a P2A, a E2A or a F2A.

Provided is a vector containing any of the provided polynucleotides. Insome cases, the vector is a viral vector. In some of any of the providedembodiments, the viral vector is a retroviral vector or a lentiviralvector.

Provided is an engineered cell containing any of the providedpolynucleotides or vectors. Also provided is an engineered cellcontaining any of the provided variant ICOSL polypeptides,immunomodulatory proteins, or fusion proteins.

Provided is an engineered cell containing any of the provided nucleicacid molecules or the vectors. In some cases, the nucleic acid encodingthe variant ICOSL polypeptide, immunomodulatory protein or fusionprotein encodes a signal peptide. In some of any of the providedembodiments, the variant ICOSL polypeptide, immunomodulatory protein orfusion protein does not contain a transmembrane domain and/or is notexpressed on the surface of the cell. In some of any of the providedembodiments, the variant ICOSL polypeptide, immunomodulatory protein orfusion protein is secreted from the engineered cell. In some aspects,the engineered cell contains a variant ICOSL polypeptide containing atransmembrane domain. In some aspects, the variant ICOSL polypeptide isexpressed on the surface of the cell.

In some of any of the provided embodiments, the cell is an immune cell.In some cases, the immune cell is an antigen presenting cell (APC) or alymphocyte. In some examples, the engineered cell is primary cell. Insome instances, the cell is a mammalian cell. In some cases, the cell isa human cell. In some of any of the provided embodiments, the lymphocyteis a T cell. In some examples, the engineered cell is an APC and the APCis an artificial APC.

In some of any of the provided embodiments, the engineered cell furthercontains a chimeric antigen receptor (CAR) or an engineered T-cellreceptor.

Provided is an infectious agent containing a nucleic acid moleculeencoding a provided variant ICOSL polypeptide or a providedimmunomodulatory protein, a provided the fusion protein. In some cases,the encoded variant ICOSL polypeptide, immunomodulatory protein orfusion protein does not contain a transmembrane domain and/or is notexpressed on the surface of a cell in which it is expressed. In some ofany of the provided embodiments, the encoded variant ICOSL polypeptide,immunomodulatory protein or fusion protein is secreted from theinfectious agent when it is expressed. In some cases, the encodedvariant ICOSL polypeptide comprises a transmembrane domain.

In some of any of the provided embodiments, the encoded variant ICOSLpolypeptide is expressed on the surface of a cell in which it isexpressed. In some cases, the infectious agent is a bacteria or a virus.In some of any of the provided embodiments, the virus is an oncolyticvirus. In some examples, the oncolytic virus is an adenoviruses,adeno-associated viruses, herpes viruses, Herpes Simplex Virus,Vesticular Stomatic virus, Reovirus, Newcastle Disease virus,parvovirus, measles virus, vesticular stomatitis virus (VSV), Coxsackievirus or a Vaccinia virus.

In some of any of the provided embodiments, the virus specificallytargets dendritic cells (DCs) and/or is dendritic cell-tropic. In somecases, the virus is a lentiviral vector that is pseudotyped with amodified Sindbis virus envelope product. In some of any of the providedembodiments, the infectious agent further contains a nucleic acidmolecule encoding a further gene product that results in death of atarget cell or that can augment or boost an immune response. In some ofany of the provided embodiments, the further gene product is selectedfrom an anticancer agent, anti-metastatic agent, an antiangiogenicagent, an immunomodulatory molecule, an immune checkpoint inhibitor, anantibody, a cytokine, a growth factor, an antigen, a cytotoxic geneproduct, a pro-apoptotic gene product, an anti-apoptotic gene product, acell matrix degradative gene, genes for tissue regeneration or areprogramming human somatic cells to pluripotency.

Provided is a pharmaceutical composition containing any of the variantICOSL polypeptides, immunomodulatory proteins, conjugates or fusionproteins or any of the provided engineered cells or infectious agents.In some of any of the provided embodiments, the pharmaceuticalcomposition contains a pharmaceutically acceptable excipient. In some ofany of the provided embodiments, the pharmaceutical composition issterile.

In some of any of the provided embodiments, provided is an article ofmanufacture containing the pharmaceutical composition in a vial. In somecases, the vial is sealed.

Provided is a kit containing any of the provided compositions andinstructions for use. Also provided is a kit containing any of theprovided articles of manufacture and instructions for use.

Provided is a method of modulating an immune response in a subject,including administering the pharmaceutical composition to the subject.Also provided is a method of modulating an immune response in a subjectincluding administering the engineered cells. In some cases, theengineered cells are autologous to the subject. In some of any of theprovided embodiments, the engineered cells are allogenic to the subject.

In some of any of the provided embodiments, the modulating the immuneresponse treats a disease or condition in the subject. In some aspects,the immune response is increased.

In some of any of the provided embodiments, an immunomodulatory proteinor conjugate containing a variant ICOSL polypeptide linked to atumor-localizing moiety is administered to the subject. In some cases,the tumor-localizing moiety is or comprises a binding molecule thatrecognizes a tumor antigen. In some of any of the provided embodiments,the binding molecule contains an antibody or an antigen-binding fragmentthereof or comprises a wild-type IgSF domain or variant thereof. In someof any of the provided embodiments, the immunomodulatory protein or theconjugate or fusion protein is administered to the subject. In some ofany of the provided embodiments, a variant ICOSL polypeptide that is atransmembrane immunomodulatory protein is administered to the subject.In some cases, the engineered cell containing a variant ICOSLpolypeptide that is a transmembrane immunomodulatory protein isadministered to the subject. In some of any of the provided embodiments,the disease or condition is a tumor or cancer. In some examples, thedisease or condition is selected from melanoma, lung cancer, bladdercancer, a hematological malignancy, liver cancer, brain cancer, renalcancer, breast cancer, pancreatic cancer, colorectal cancer, spleencancer, prostate cancer, testicular cancer, ovarian cancer, uterinecancer, gastric carcinoma, a musculoskeletal cancer, a head and neckcancer, a gastrointestinal cancer, a germ cell cancer, or an endocrineand neuroendocrine cancer. In some of any of the provided embodiments,the immune response is decreased.

In some of any of the provided embodiments, a variant ICOSL polypeptideor immunomodulatory protein that is soluble is administered to thesubject. In some of any of the provided embodiments, the solubleimmunomodulatory protein is an immunomodulatory Fc fusion protein. Insome of any of the provided embodiments, a provided variant ICOSLpolypeptide, immunomodulatory protein, or fusion protein is administeredto the subject. In some of any of the provided embodiments, anengineered cell containing a secretable variant ICOSL polypeptide isadministered to the subject. In some of any of the provided embodiments,a provided engineered cell is administered to the subject. In some ofany of the provided embodiments, an infectious agent encoding a variantICOSL polypeptide that is a secretable immunomodulatory protein isadministered to the subject, optionally under conditions in which theinfectious agent infects a tumor cell or immune cell and the secretableimmunomodulatory protein is secreted from the infected cell. In some ofany of the provided embodiments, the disease or condition is aninflammatory or autoimmune disease or condition. In some examples, thedisease or condition is an Antineutrophil cytoplasmic antibodies(ANCA)-associated vasculitis, a vasculitis, an autoimmune skin disease,transplantation, a Rheumatic disease, an inflammatory gastrointestinaldisease, an inflammatory eye disease, an inflammatory neurologicaldisease, an inflammatory pulmonary disease, an inflammatory endocrinedisease, or an autoimmune hematological disease. In some cases, thedisease or condition is selected from inflammatory bowel disease,transplant, Crohn's disease, ulcerative colitis, multiple sclerosis,asthma, rheumatoid arthritis, or psoriasis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts impedance results reflecting cytotoxic killing activityof cells engineered with an anti-CD19 chimeric antigen receptor (CAR)alone or with an exemplary transmembrane immunomodulatory TIP (CD80-TIPor ICOSL-TIP) or the corresponding CD80 or ICOSL wild-type transmembraneprotein following co-culture with target antigen-expressing cells.Impedance was assessed using the Acea Real-Time Cell Analyzer (RTCA),which measures the impedance variations in the culture media of a96-well microelectronic plate (E-plate).

FIG. 2A depicts that primary T cells are effectively transduced withviruses encoding both the CAR and TIP proteins. Primary human T cellsactivated 48 hours with anti-CD3 plus anti-CD28 beads and were thentranduced with a Lenti-virus encoding an anti-CD19 CAR with a BFPreporter, plus a second Lenti-virus encoding and ICOSL TIP with a GFPreporter. The FACs plot shows BFP expression on the y-axis and GFPexpression on the x-axis and the percentage of T cells that fall intoeach quadrant are indicated. Results show that the cultures include CARonly transduced cells (upper left quadrant), TIP only transduced cells(lower right quadrant), cells transduced with both viruses (upper rightquadrant) and cells that were not transduced with either (lower left).In FIG. 2B, TIPs expressed on CAR-T cells provide costimulation to theCAR-T cells. CAR-T cells with or without TIP co-transduction werelabeled with Cell-Trace Far Red and incubated with the CD19+ NALM6 cellline to engage the CAR. Proliferation was assessed by the percentage ofCAR-expressing cells that had diluted out the fluorescent dye. Cellstransduced with mutated TIPs showed an increase proliferation of CAR+ Tcells compared to those without TIPs or those transduced with wild-typeICOSL. Mock transduced cells that lacked CAR expression failed toproliferate in this assay.

FIG. 3A-3B demonstrate, via cytokine release, the costimulatory capacityof wild-type (WT) or variant ICOSL when coimmobilized with anti-CD3. 10nM anti-CD3 was wet coated to the wells of 96-well flat bottomedpolystyrene tissue culture plates with 40 nM (arrows) or 10 nM WT orvariant ICOSL. 100,000 purified CD4⁺ and CD8⁺ (pan) T-cells cells wereadded and supernatant was harvested 72 hours later for ELISA analysisfor cytokine release. FIG. 3A shows IFN-gamma and FIG. 3B shows IL-17protein levels secreted from pan T-cells. Graphs are representative oftypical IFN-gamma and IL-17 responses from pan T-cell costimulation.

FIG. 4A-4B demonstrate, via proliferation, the costimulatory capacity ofwild-type (WT) or variant ICOSL when coimmobilized with anti-CD3.CFSE-labeled pan T-cells were incubated in anti-CD3 and ICOSL coatedplates as previously described for 72 hours. Cells were harvested,washed, stained with fluorescently conjugated anti-CD4 or anti-CD8antibodies, and analyzed by flow cytometry. Gates and cytometer voltageswere set using non-stimulated control CFSE-labeled T-cells.Proliferation was determined by CFSE dilution from control. FIG. 4Ashows percent of total proliferating (arrows), CD4⁺ (solid bar), andCD8⁺ cells (hatched bar) T-cells following 40 nM ICOSL costimulation.FIG. 4B shows percent of total pan T-cell proliferation following 10 nMICOSL costimulation. Graphs are representative of typical proliferativeresponse from pan T-cell costimulation.

FIG. 5 depicts ICOSL vIgD candidate function in a humanMixed-Lymphocyte-Reation (MLR). ICOSL variants and their mutations arelisted on the x-axis, along with wild-type ICOSL, negative controlsPDL2-Fc and human IgG, as well as the positive control benchmarkmolecule CTLA-Ig Belatacept. The line across the graph represents thebaseline amount of IFN-gamma detected in the supernatants of negativecontrol cultures. For each ICOSL variant candidate or control, threedifferent concentrations were tested with arrows indicating the highestconcentration of protein in cultures at 40 nM. The majority of ICOSLvariant candidates show superior antagonistic activity at all threeconcentrations tested compared to belatacept as reflected by the lowerconcentration of IFN-gamma in those cultures.

FIG. 6A-6D depicts the inhibition of soluble ICOSL Fc-fusion proteins onB and T cell responses in a B-T co-culture assay. FIG. 6A depictssoluble ICOSL Fc-fusion proteins inhibition of T cell-driven B cellproliferation. Purified CD4+ T cells and B cells from a single donorwere CFSE-labeled and co-incubated at a 1:1 ration in the presence orabsence of the indicated mitogens with or without the indicated ICOSLFc-fusion proteins. Cells were stimulated with Staph enterotoxin B (SEB)at 100 ng/mL, Pokeweed mitogen (PWM) at 1 mg/mL, or both. ICOSLFc-fusion proteins were included at a final concentration of 40 nM andcultures were incubated for 7 days and subjected to FACS analysis. Thenumber of divided B cells was determined from the number of cells in thecultures that had diluted their CFSE. All of the ICOSL Fc-fusionproteins tested except for wild-type reduced B cell proliferation. FIG.6B-6D show ICOSL Fc-fusion proteins inhibited cytokine T cell cytokineproduction in B-T co-cultures. Supernatants from the cultures describedabove were harvested on day 7 and analyzed for cytokine content using aLEGENDplex Human Th Cytokine Panel (Biolegend). T cell production ofIL-5 (FIG. 6B), IL-13 (FIG. 6C) and IL-21 (FIG. 6D) is attenuated byinclusion of ICOSL Fc-fusion proteins.

FIG. 7A-7F depicts different endpoints in a mouse model of Graft VerseHost Disease (GVHD) where human PBMC cells were adoptively transferredinto immunodeficient NSG murine hosts. FIG. 7A shows survival curves ofthe treated animals. Aggressive disease course and subsequent mortalitywas observed in the saline control animals, with similar survivalobserved in the animals treated with wild-type ICOSL-Fc, as well as theN52H/I143T ICOSL variant. Variant N52H/N57Y/Q100P had improved survivalrates comparable to the clinical benchmark belatacept. FIG. 7B showssimilar trends in body weight loss, with ICOSL variant N52H/N57Y/Q100Pdemonstrating similar weight maintenance as animals treated withbelatacept, even though all other groups experienced rapid weight loss.FIG. 7C shows clinical scores from standardized GVHD Disease ActivityIndex (DAI) observations, again showing lower scores in animals treatedwith the ICOSL variant N52H/N57Y/Q100P that are comparable to theclinical benchmark belatacept while the other groups of animalsexperienced higher DAI scores. FIG. 7D depicts a flow cytometricmeasurement of CD4 and CD8 percentages in blood from experimentalanimals measured on day 14. The percentage of CD8 cells betweenexperimental groups was largely the same, however, animals treated withICOSL variant N52H/N57Y/Q100P and belatacept have lower percentages ofCD4 cells compared to the other experimental groups.

FIG. 7E depicts survival curves from a similar experiment testingadditional ICOSL variant molecules. FIG. 7F depicts clinical scores froma similar experiment testing additional ICOSL variant molecules.

FIG. 8 shows localized costimulatory activity conveyed by the indicatedvariant stack molecule vIgD C-L, where C represents an ICOSLcostimulatory domain and L represents a NKp30 localizing domain. In thisassay, target K562 cells expressing the localizing surface protein,B7-H6, were cultured in the presence of anti-CD3 with human T cells andT cell activation was assessed by IFN-gamma levels in culturesupernatants. Including anti-CD3 alone or no stack variant Fc moleculesdid not induce T cell activation. Similarly, cells cultured with onlythe wild-type localizing NKp30 domain alone or the wild-typecostimulatory ICOSL domain alone as Fc fusion proteins did not result inT cell activation. A stacked domain containing the wild-type version ofboth the costimulatory domain and localizing domain induced measurableIFN-gamma at the highest concentration tested, however, the variantlocalizing costimulatory stack induced greater than two fold higherIFN-gamma levels at the highest concentration, and IFN-gamma levels thatwere still observed as the concentrations were titrated down.

FIG. 9 summarizes changes in ear thickness in mice from a standard modelof Delayed-Type Hypersensitivity (DTH). PBS treated animals sensitizedwith ovalbumin and subsequently challenged in the ear with the sameprotein, show the highest level of measured ear swelling. Mice treatedwith clinical benchmark Abatacept have slightly reduced ear swellingfollowing ear challenge. All five ICOSL variant treatment groupsdemonstrated equal or improved reductions in ear swelling compared toAbatacept.

FIG. 10A-10C depicts various exemplary configurations of a variant IgSFdomain (vIgD) conjugated to an antibody (V-Mab). FIG. 10A shows variousconfigurations in which a vIgD is linked, directly or indirectly, to theN- and/or C-terminus of the light chain of an antibody. FIG. 10B showsvarious configurations in which a vIgD is linked, directly orindirectly, to the N- and/or C-terminus of the heavy chain of anantibody. FIG. 10C depicts the resulting V-Mab configurations when alight chain of FIG. 10A and a heavy chain of FIG. 10B are co-expressedin a cell.

FIG. 11A-11B demonstrate V-Mab specificity for cognate binding partners.Binding assays were performed on Expi293 cells transiently transfectedwith DNA for mammalian surface expression of human HER2, CD28, CTLA-4,or ICOS. 200,000 transfected cells were incubated with 100,000 pM to 100pM parental antibody (C1) or various V-Mabs (C2-9). Unbound antibody wasremoved, bound antibody detected with fluorescently conjugatedanti-human IgG, and the cells were analyzed by flow cytometry for MFIand percentage positive based on Fc controls. FIG. 11A shows binding ofthe V-Mabs to HER2 transfectants at levels similar to the parentalantibody. Binding to mock transfected cells is observed with all V-Mabs,though not WT ICOSL, due to low levels of endogenous HER2 expression onExpi293 parental cells. FIG. 11B shows binding of the parentalIgSF-domain (N52H/N57Y/Q100P) to its cognate partners is maintained orincreased (C2, C3, C4, C5, C6, C8, C9) by V-Mabs.

FIG. 12 demonstrates V-Mab costimulatory and proliferative capacity whencoimmobilized with anti-CD3. 10 nM anti-CD3 was wet coated to the wellsof 96-well flat bottomed polystyrene tissue culture plates with 30 nM to3 nM parental antibody, V-Mabs, or Fc controls. CFSE-labeled pan T-cellswere added for 72 hours. IFN-gamma secretion was measured by ELISA andtotal T-cell proliferation was measured by flow cytometric analysis ofCFSE-dilution. IFN-gamma secretion and proliferation of IgSF-domain(N52H/N57Y/Q100P) is greater than WT ICOSL. V-Mabs demonstrate increasedcytokine and proliferative costimulatory capacity over the parentalIgSF.

FIG. 13A-13C depicts various formats of the provided variant IgSF domainmolecules. FIG. 13A depicts soluble molecules, including: (1) a variantIgSF domain (vIgD) fused to an Fc chain; (2) a stack molecule containinga first variant IgSF domain (first vIgD) and a second IgSF domain, suchas a second variant IgSF domain (second vIgD); (3) a tumor targetingIgSF molecule containing a first variant IgSF domain (vIgD) and an IgSFdomain that targets to a tumor antigen, such as an NKp30 IgSF domain;and (4) a variant IgSF domain (vIgD) linked to an antibody (V-Mab). FIG.13B depicts a transmembrane immunomodulatory protein (TIP) containing avariant IgSF domain (vIgD), e.g., variant ICOSL, expressed on thesurface of a cell. In an exemplary embodiment, the cognate bindingpartner of the transmembrane bound vIgD is a costimulatory receptor,e.g. CD28, and the TIP containing the vIgD (e.g. ICOSL vIgD) agonizesthe costimulatory receptor such that the TIP induces a positive signalin the cell expressing the costimulatory receptor. FIG. 13C depicts asecreted immunomodulatory protein (SIP) in which a variant IgSF domain(vIgD), e.g., variant ICOSL, is secreted from a cell, such as a first Tcell (e.g. CAR T cell). In an exemplary embodiment, the cognate bindingpartner of the secreted vIgD is an activating receptor, e.g., CD28,which can be expressed on the first cell (e.g., T cell, such as a CAR Tcell) and/or on a second cell (e.g. T cell; either endogenous orengineered, such as a CAR T cell). Upon binding of the SIP with itscognate binding partner, signaling via the activating receptor isblocked. In all cases, the vIgD can be a V-domain (IgV) only, thecombination of the V-domain (IgV) and C-domain (IgC), including theentire extracellular domain (ECD), or any combination of Ig domains ofthe IgSF superfamily member.

FIG. 14 depicts an exemplary schematic of the activity of a variant IgSFdomain (vIgD) fused to an Fc (vIgD-Fc) in which the vIgD is a variant ofan IgSF domain of ICOSL. As shown, a soluble vIgD of ICOSL interactswith its cognate binding partners to block interactions of CD80(B7-1)/CD86 (B7-2) or ICOSL with CD28 or ICOS, respectively, therebyblocking costimulation by the CD28 and/or ICOS costimulatory receptors.

FIG. 15 depicts an exemplary schematic of a stack molecule forlocalizing the variant IgSF domain (vIgD) to a tumor cell. In thisformat, the stack molecule contains a first variant IgSF domain (firstvIgD) and a second IgSF domain (e.g. a second vIgD) in which the secondIgSF domain (e.g. a second vIgD) is a tumor-targeted IgSF domain thatbinds to a tumor antigen. An exemplary tumor-targeted IgSF domain is anIgSF domain of NKp30, which binds to the tumor antigen B7-H6. In thisdepiction, the vIgD is a variant of an IgSF domain of ICOSL. As shown,binding of tumor-targeted IgSF domain to the surface of the tumor celllocalizes the first vIgD on the tumor cell surface where it can interactwith one or more of its cognate binding partners (e.g. CD28 or ICOS)expressed on the surface of an adjacent immune cell (e.g. T cell) tostimulate the costimulatory receptor.

FIG. 16A depicts various exemplary configurations of a stack moleculecontaining a first variant IgSF domain (first vIgD), e.g. variant ICOSL,and a second IgSF domain, such as a second variant IgSF domain (secondvIgD). As shown, the first vIgD and second IgSF domain are independentlylinked, directly or indirectly, to the N- or C-terminus of an Fc region.For generating a homodimeric Fc molecule, the Fc region is one that iscapable of forming a homodimer with a matched Fc subunit byco-expression of the individual Fc regions in a cell. For generating aheterodimeric Fc molecule, the individual Fc regions contain mutations(e.g. “knob-into-hole” mutations in the CH3 domain), such that formationof the heterodimer is favored compared to homodimers when the individualFc regions are co-expressed in a cell.

FIG. 16B depicts various exemplary configurations of a stack moleculecontaining a first variant IgSF domain (first vIgD), a second IgSFdomain, such as a second variant IgSF domain (second vIgD), and a thirdIgSF domain, such as a third variant IgSF domain (third vIgD). As shown,the first vIgD, second IgSF, and third IgSF domains are independentlylinked, directly or indirectly, to the N- or C-terminus of an Fc region.For generating a homodimeric Fc molecule, the Fc region is one that iscapable of forming a homodimer with a matched Fc region by co-expressionof the individual Fc regions in a cell.

FIG. 17 depicts an exemplary schematic of the activity of a variant IgSFdomain (vIgD) conjugated to an antibody (V-Mab) in which the antibody(e.g. anti-HER2 antibody) binds to an antigen on the surface of thetumor cell. In this depiction, the vIgD is a variant of an IgSF domainof ICOSL. As shown, binding of the antibody to the surface of the tumorcell localizes the vIgD on the tumor cell surface where it can interactwith one or more of its cognate binding partners expressed on thesurface of an adjacent immune cell (e.g. T cell) to agonize receptorsignaling. In an exemplary embodiment as shown, the variant IgSF domain(vIgD) is a variant of an IgSF domain of ICOSL. Binding of the ICOSLvIgD to CD28 or ICOS costimulatory receptors provides an agonist orcostimulatory signal.

FIG. 18 depicts the Nanostring transcriptional signature of primaryhuman T cells when incubated 10 nM anti-CD3 with 40 nM of an Fc-controlprotein, wild-type ICOSL-Fc, wild-type CD80-Fc, both of these proteins,or a variant ICOSL Fc-fusion proteins with mutations as indicated. TotalRNA from samples was prepared from harvested cells and the RNA wastransferred to Nanostring and a Cancer Immune chip was used toquantitate transcripts of 750 gene in each sample. Altered transcriptsinclude those whose level is above or below the diagonal line, includingthe noted transcripts.

FIG. 19 depicts transcript levels of exemplary transcripts uponincubation as described in FIG. 18 for the indicated times in thepresence of the various immunomodulatory proteins.

FIG. 20A-20B demonstrates VmAb mediated T-cell proliferation whenco-cultured with HER2 expressing targets. CFSE-labeled pan T-cells wereactivated with K562-derived artificial target cells displaying cellsurface anti-CD3 single chain Fv (OKT3) and HER2 in the presence ofVmAbs or control proteins. Proliferation was measured by flow cytometricanalysis of CFSE-dilution on CD4⁺ (left panel) or CD8⁺ (right panel)stained T-cells. In FIG. 20A, K562 cells were titrated and plated withT-cells for an effector:target (E:T) ratio of 40 to 1280:1. VmAbs,parental IgSF domain, or WT ICOSL were added at 1000 pM. In FIG. 20B,K562 cells were added to T-cells for an E:T ratio of 160:1. VmAbs orcontrol proteins were titrated and added at 3000 to 37 pM.

FIG. 21 depicts the proliferation studies for T cells transduced withvarious IgSF domain-containing transmembrane immunomodulatory proteins(TIPs) and an exemplary recombinant E6-specific TCRs in primary human Tcells.

FIG. 22A-22G shows SEC analysis of proteolysis in variant ICOSLFc-fusion molecules containing mutations N52H/N57Y/Q100R/F172S generatedin various reference sequences, such as truncated ICOSL ECD Fc-fusion,an ICOSL IgV domain alone Fc-fusion, and/or ICOSL variant Fc fusionproteins with mutations at N207G/L208G with reference to the referenceICOSL extracellular domain (ECD) sequence set forth in SEQ ID NO:32.Molecules were expressed using ExpiCHO-S derived cells.

FIG. 23A-23B depicts the proliferation of CD4 and CD8 human T cellsstimulated with K652 cells expressing variant ICOSL TIPs containing anECD containing an affinity-modified IgSF with amino acid mutationscorresponding to N52H/N57Y/Q100P (SEQ ID NO: 288), N52H/N57Y/Q100R (SEQID NO: 283), and E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R (SEQID NO: 300).

FIG. 24A depicts binding of V-mAbs to HER2 and CD28. FIG. 24B-24Fdepicts results of VmAb costimulation of T cells using a transfectedcell system using Jurkat cells with an IL-2 promoter luciferasereporter, showing that V-mAbs provided a significant costimulatorysignal in the presence of HER2+K562/OKT3 cells.

FIG. 25A-25D depicts binding of stack Fc-fusion molecules to cellsexpressing cognate binding partners B7H6 (FIG. 25A), ICOS (FIG. 25B),CD28 (FIG. 25C), and CTLA-4 (FIG. 25D).

FIG. 26A-26B depicts bioactivity studies for exemplary testedICOSL/NKp30 stack proteins.

FIG. 27 depicts proliferation induced by ICOSL/NKp30 stack proteins asmeasured by flow cytometric analysis of CFSE-dilution on CD4+ or CD8+stained T-cells.

FIG. 28 depicts anti-tumor effects of the combination of the testedICOSL/NKp30 stack protein and mPD-1 mAb.

FIG. 29A-29E depicts anti-inflammatory activity of prophylactic dosingof the exemplary ICOSL IgV-Fc fusion molecule in the collagen-inducedarthritis (CIA) model, including mean sum paw score (FIG. 29A), detectedCII IgG (FIG. 29B), serum cytokine levels (FIG. 29C), CD44+ activated Tcells or T_(FH) cells (FIG. 29D), and fraction of B cells in thedraining lymph node (FIG. 29E).

FIG. 30A-30D depicts anti-inflammatory activity of delayed dosing of theexemplary ICOSL IgV-Fc fusion molecule in the collagen-induced arthritis(CIA) model, including mean sum paw score (FIG. 30A) and serum cytokinelevels (FIG. 30C-30D).

FIG. 31A-31D depicts anti-inflammatory activity of delayed dosing of theexemplary ICOSL IgV-Fc fusion molecule in the experimental autoimmuneencephalomyelitis (EAE) model, including EAE score (FIG. 31A), flowcytometric analysis of inguinal lymph node T cells (FIG. 31C), andproinflammatory cytokines (FIG. 31D).

FIG. 32A-32B depicts survival and DAI score of Graft-versus-Host-Disease(GvHD) mice treated with various doses (20, 100, or 500 μg) of a variantICOSL IgV-Fc molecule.

FIG. 33A-33F depicts results from flow cytometric analysis ofGraft-versus-Host-Disease (GvHD) ratio of human cells/mouse cells inblood collected (FIG. 33A) or in total T cell count (FIG. 33B) at theend of the study, and assessment of ICOS+CD4+ or CD8+ cells (FIG.33C-33D), or CD28+CD4+ or CD8+ cells (FIG. 33E-33F) fromGraft-versus-Host-Disease (GvHD) mice treated with various doses (20,100, or 500 μg) of a variant ICOSL IgV-Fc molecule.

FIG. 34A-34B depicts expression of activation or exhaustion markers of Tcells from Graft-versus-Host-Disease (GvHD) mice treated with variousdoses (20, 100, or 500 μg) of a variant ICOSL IgV-Fc molecule.

FIG. 34C depicts the ratio of T effector cells (Teff) to T regulatorycells (Treg) from Graft-versus-Host-Disease (GvHD) mice treated withvarious doses (20, 100, or 500 μg) of a variant ICOSL IgV-Fc molecule.

FIG. 35A-35D depicts serum proinflammatory cytokines fromGraft-versus-Host-Disease (GvHD) mice treated with various doses (20,100, or 500 μg) of a variant ICOSL IgV-Fc molecule. FIG. 35E depictsserum exposure of variant ICOSL IgV-Fc (N52H/N57Y/Q100R) in the GVHDmodel compared to normal mice.

FIG. 36A depicts DAI results and FIG. 36B depicts histology results fromtreatment with an exemplary variant ICOSL IgV-Fc on disease activityindex (DAI) calculated from body weight and stool scores in aCD4+CD45RBhigh-induced colitis model.

DETAILED DESCRIPTION

Provided herein are immunomodulatory proteins that are or comprisevariants or mutants of ICOS ligand (ICOSL) or specific binding fragmentsthereof that exhibit activity to bind to at least one target ligandcognate binding partner (also called counter-structure protein). In someembodiments, the variant ICOSL polypeptides contain one or more aminoacid modifications (e.g. amino acid substitutions, deletions oradditions) compared to a reference (e.g., unmodified) or wild-type ICOSLpolypeptide. In some embodiments, the one or more amino acidmodifications (e.g. amino acid substitutions, deletions or additions)are in an immunoglobulin superfamily (IgSF) domain (e.g. IgV) of areference (e.g., unmodified) or wild-type ICOSL polypeptide. In someembodiments, the variant ICOSL polypeptide exhibits altered, such asincreased or decreased, binding activity or affinity for at least onecognate binding partner, such as at least one of ICOS, CD28, or CTLA-4.In some embodiments, the immunomodulatory proteins are soluble. In someembodiments, the immunomodulatory proteins are transmembraneimmunomodulatory proteins capable of being expressed on the surface ofcells. In some embodiments, also provided herein are one or more otherimmunomodulatory proteins that are conjugates or fusions containing avariant ICOSL polypeptide provided herein and one or more other moietyor polypeptide.

In some embodiments, the variant ICOSL polypeptides and immunomodulatoryproteins modulate an immunological immune response, such as an increasedor decreased immune response. In some embodiments, the variant ICOSLpolypeptides and immunomodulatory proteins provided herein can be usedfor the treatment of diseases or conditions that are associated with adysregulated immune response.

In some embodiments, the provided variant ICOSL polypeptides modulate Tcell activation via interactions with costimulatory signaling molecules.In general, antigen specific T-cell activation requires two distinctsignals. The first signal is provided by the interaction of the T-cellreceptor (TCR) with major histocompatibility complex (MHC) associatedantigens present on antigen presenting cells (APCs). The second signalis costimulatory to TCR engagement and necessary to avoid T-cellapoptosis or anergy.

In some embodiments, under normal physiological conditions, the Tcell-mediated immune response is initiated by antigen recognition by theT cell receptor (TCR) and is regulated by a balance of co-stimulatoryand inhibitory signals (e.g., immune checkpoint receptors). The immunesystem relies on immune checkpoint receptors to prevent autoimmunity(i.e., self-tolerance) and to protect tissues from excessive damageduring an immune response, for example during an attack against apathogenic infection. In some cases, however, these immunomodulatoryproteins can be dysregulated in diseases and conditions, includingtumors, as a mechanism for evading the immune system.

In some embodiments, among known T-cell costimulatory receptors is CD28,which is the T-cell costimulatory receptor for the ligands B7-1 (CD80)and B7-2 (CD86) both of which are present on APCs. These same ligandscan also bind to the inhibitory T-cell receptor CTLA4 (cytotoxicT-lymphocyte-associated protein 4) with greater affinity than for CD28;the binding to CTLA-4 acts to down-modulate the immune response. ICOS(inducible costimulator) is another T-cell costimulatory receptor whichbinds to ICOS ligand (ICOSL) on APCs. In some cases, CD28 and CTLA-4also are known to interact with ICOSL at a binding site that overlapswith the binding of ICOSL to the T-cell costimulatory receptor ICOS (Yaoet al. (2011) Immunity, 34:729-740). Although CD28 and ICOS are relatedCD28 family activating receptors and share some intracellular signalingmotifs, costimulatory effects between CD28 and ICOS differ. For example,CD28 is expressed on both unactivated and activated T cells and itssignaling is important for IL-2 production and subsequent T celleffector function. ICOS is generally not expressed on the surface of Tcells until after T cell activation, and signaling through ICOS onactivated T cells supports specialized T cell subset differentiation.Thus, in some cases, costimulation by CD28 and ICOS yields overlappingand complementary effects.

In some aspects, T cells express the costimulatory molecules CD28 andICOS, which interact with CD80/CD86 and ICOSL respectively, on antigenpresenting cells (APC). In lymphoid organs, professional APC (i.e.dendritic cells, macrophages, and B cells) express CD80, CD86, and ICOSLand engage CD28+/ICOS+ T cells. In some embodiments, activated T cellscan then differentiate into effector cells such as CD8+ cytotoxic Tcells (CTL), IL-17A/F-secreting CD4+Th17 cells, or CD4+ follicularhelper (T_(FH)) cells. T_(FH)-expressing CD40L engage B cells inlymphoid follicles and release cytokines (e.g. IL-21) inducingdifferentiation of B cells to antibody (Ab)-secreting plasma cells.Plasma cells can produce tissue-damaging antibodies, e.g., rheumatoidfactor (RF) and anti-citrullinated peptide antibodies (ACPA) in humans,and anti-collagen (CII) antibodies in mice, which can form immunecomplexes and deposits in the joints and other tissues. ICOSL can alsobe expressed on non-professional APCs, leading to T cell activation innon-lymphoid tissues and further damage to the tissues and joints.

In some aspects, CD4+Th1-, Th9- and Th17-cells, are implicated as keycontributors to multiple sclerosis (MS) by increasing inflammationwithin the CNS in both multiple sclerosis and experimental autoimmuneencephalomyelitis and CD4+ ICOS+CXCR5+T follicular helper cells areincreased in PBMC in relapsing-remitting and correlate with diseaseprogression in secondary progressive MS. In some embodiments, there issignificantly increased ICOS gene expression in cerebrospinal fluidcells, in secondary progressive MS, and an increased percentage of totalmonocytes and monocytes expressing ICOSL is observed. ICOSL alsoexpressed on non-professional APCs, leading to T cell activation innon-lymphoid tissues and further tissue damage

Among the provided variant ICOSL polypeptide are polypeptides that, whenmodified by one or more amino acid modifications of an IgSF domain of areference ICOSL polypeptide, exhibit enhanced binding affinity for CD28and/or ICOS. In some cases, the overall increase in ICOS binding inprovided variants is less than the increase in CD28 binding becausewild-type ICOSL already demonstrates substantially more binding affinityfor ICOS than CD28. Also provided are various formats of the providedvariant polypeptides. As shown herein, alternative formats canfacilitate manipulation of the immune response, and hence thetherapeutic application. For example, delivery of enhanced ICOSLproteins in soluble formats is shown herein to antagonize T cellactivation by inhibiting CD28 and/or ICOS signaling. In other examples,tethering of the variant ICOSL molecules to a surface facilitates T cellactivation by providing a costimulatory signal. Various tetheringstrategies are provided to localize delivery of a T cell costimulatorysignal including, but not limited to, direct coating to plastic, use ofanother variant IgSF domain to localize to a plate-bound or cell surfaceexpressed protein target, or fusion of the variant ICOSL to atumor-specific monoclonal antibody.

In some embodiments, the modulation of immune signaling achieved by theprovided variant ICOSL polypeptides and immunomodulatory polypeptidesoffers advantages for treatment of inflammatory and autoimmune disordersand other diseases and conditions compared to other treatments. In somecases, therapies to intervene and alter the costimulatory effects ofboth receptors are constrained by the spatial orientation requirementsas well as size limitations imposed by the confines of the immunologicalsynapse. In some aspects, existing therapeutic drugs, including antibodydrugs, may not be able to interact simultaneously with the multipletarget proteins involved in modulating these interactions. In addition,in some cases, existing therapeutic drugs may only have the ability toantagonize but not agonize an immune response. Additionally,pharmacokinetic differences between drugs that independently target oneor the other of these two receptors can create difficulties in properlymaintaining a desired blood concentration of such drug combinationsthroughout the course of treatment.

In some embodiments, the provided variant ICOSL polypeptides orimmunomodulatory proteins modulate (e.g. increase or decrease)immunological activity induced by costimulatory receptors CD28 or ICOS.Thus, in some embodiments, the provided polypeptides overcome theseconstraints by providing variant ICOSL (inducible costimulator ligand)with altered (e.g. increased or decreased) binding affinities to bothCD28 and ICOS, and, in some cases, CTLA-4, thereby agonizing orantagonizing the complementary effects of costimulation by receptors.Methods of making and using these variant ICOSL are also provided.

In some aspects, the provided molecules may also be more effective thanother soluble therapeutic protein agents. For example, abatacept(CTLA-4-Fc) has been shown to interfere with T cell costimulation toattenuate T cell responses in autoimmune disease settings, such as forthe treatment of rheumatoid arthritis, psoriatic arthritis and juvenileidiopathic arthritis, and belatacept, a variant CTLA-4-Fc molecule, fortransplant rejection. These CTLA-4-Fc proteins, however, bind to CD80and CD86 and prevent these costimulatory ligands from engaging andtriggering only CD28. Variant ICOSL polypeptides provided herein, insome cases, exhibit binding affinity and enhanced activity for both CD28and ICOS.

Furthermore, the ability to format the variant polypeptides in variousconfigurations to, depending on the context, antagonize or agonize animmune response, offers flexibility in therapeutic applications based onthe same increased binding and activity of a variant ICOSL for bindingpartners. In some embodiments, the particular format can be chosen forthe desired therapeutic application. For example, as described, animmunomodulatory polypeptide comprising a variant ICOSL polypeptide isprovided in a format, e.g. as an Fc-fusion protein, to antagonize orblock activity of its cognate binding partner, e.g. ICOS and/or CD28. Insome embodiments, blocking or inhibiting costimulatory signaling viaCD28 or ICOS may be useful to suppresses an immune response, which canbe useful in the treatment of inflammatory or autoimmune disorders(e.g., multiple sclerosis or brain inflammation), or organtransplantation. As an example, tethering variant ICOSL proteins to asurface can deliver a localized costimulatory signal, which, in someaspects, can be used to target tumor tissue to deliver localizedcostimulation to tumor infiltrating T cells. Most primary tumors lackexpression of costimulatory molecules such as CD80, CD86 or ICOSL, andthus T cell anti-tumor responses can be compromised by a lack ofcostimulation (Yu et al. (1998) Int. Immunol. 10:791-797). By localizingcostimulatory domains to tumor cells using a tumor-localizing moiety,such as Nkp30 localized to B7H6 tumor cells or a tumor-specificantibody, T cell responses can be enhanced in the absence oftumor-expressed costimulatory proteins.

All publications, including patents, patent applications scientificarticles and databases, mentioned in this specification are hereinincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, including patent, patentapplication, scientific article or database, were specifically andindividually indicated to be incorporated by reference. If a definitionset forth herein is contrary to or otherwise inconsistent with adefinition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth herein prevails over the definitionthat is incorporated herein by reference.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

I. DEFINITIONS

Unless defined otherwise, all terms of art, notations and othertechnical and scientific terms or terminology used herein are intendedto have the same meaning as is commonly understood by one of ordinaryskill in the art to which the claimed subject matter pertains. In somecases, terms with commonly understood meanings are defined herein forclarity and/or for ready reference, and the inclusion of suchdefinitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art.

The terms used throughout this specification are defined as followsunless otherwise limited in specific instances. As used in thespecification and the appended claims, the singular forms “a,” “an,” and“the” include plural referents unless the context clearly dictatesotherwise. Unless defined otherwise, all technical and scientific terms,acronyms, and abbreviations used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which theinvention pertains. Unless indicated otherwise, abbreviations andsymbols for chemical and biochemical names is per IUPAC-IUBnomenclature. Unless indicated otherwise, all numerical ranges areinclusive of the values defining the range as well as all integer valuesin-between.

The term “affinity modified” as used in the context of an immunoglobulinsuperfamily domain, means a mammalian immunoglobulin superfamily (IgSF)domain having an altered amino acid sequence (relative to thecorresponding wild-type parental or unmodified IgSF domain) such that ithas an increased or decreased binding affinity or avidity to at leastone of its cognate binding partners (alternatively “counter-structures”)compared to the parental wild-type or unmodified (i.e., non-affinitymodified) IgSF control domain. Included in this context is an affinitymodified ICOSL IgSF domain. In some embodiments, the affinity-modifiedIgSF domain can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or moreamino acid differences, such as amino acid substitutions, in a reference(e.g., unmodified) or wild-type IgSF domain. An increase or decrease inbinding affinity or avidity can be determined using well known bindingassays such as flow cytometry. Larsen et al., American Journal ofTransplantation, Vol 5: 443-453 (2005). See also, Linsley et al.,Immunity, Vol 1: 793-801 (1994). An increase in a protein's bindingaffinity or avidity to its cognate binding partner(s) is to a value atleast 10% greater than that of the wild-type IgSF domain control and insome embodiments, at least 20%, 30%, 40%, 50%, 100%, 200%, 300%, 500%,1000%, 5000%, or 10000% greater than that of the wild-type IgSF domaincontrol value. A decrease in a protein's binding affinity or avidity toat least one of its cognate binding partner is to a value no greaterthan 90% of the control but no less than 10% of the wild-type IgSFdomain control value, and in some embodiments no greater than 80%, 70%60%, 50%, 40%, 30%, or 20% but no less than 10% of the wild-type IgSFdomain control value. An affinity-modified protein is altered in primaryamino acid sequence by substitution, addition, or deletion of amino acidresidues. The term “affinity modified IgSF domain” is not be construedas imposing any condition for any particular starting composition ormethod by which the affinity-modified IgSF domain was created. Thus, theaffinity modified IgSF domains of the present invention are not limitedto wild type IgSF domains that are then transformed to an affinitymodified IgSF domain by any particular process of affinity modification.An affinity modified IgSF domain polypeptide can, for example, begenerated starting from wild type mammalian IgSF domain sequenceinformation, then modeled in silico for binding to its cognate bindingpartner, and finally recombinantly or chemically synthesized to yieldthe affinity modified IgSF domain composition of matter. In but onealternative example, an affinity modified IgSF domain can be created bysite-directed mutagenesis of a wild-type IgSF domain. Thus, affinitymodified IgSF domain denotes a product and not necessarily a productproduced by any given process. A variety of techniques includingrecombinant methods, chemical synthesis, or combinations thereof, may beemployed.

The term “allogeneic” as used herein means a cell or tissue that isremoved from one organism and then infused or adoptively transferredinto a genetically dissimilar organism of the same species. In someembodiments of the invention, the species is murine or human.

The term “autologous” as used herein means a cell or tissue that isremoved from the same organism to which it is later infused oradoptively transferred. An autologous cell or tissue can be altered by,for example, recombinant DNA methodologies, such that it is no longergenetically identical to the native cell or native tissue which isremoved from the organism. For example, a native autologous T-cell canbe genetically engineered by recombinant DNA techniques to become anautologous engineered cell expressing a transmembrane immunomodulatoryprotein and/or chimeric antigen receptor (CAR), which in some casesinvolves engineering a T-cell or TIL (tumor infiltrating lymphocyte).The engineered cells are then infused into a patient from which thenative T-cell was isolated. In some embodiments, the organism is humanor murine.

The terms “binding affinity,” and “binding avidity” as used herein meansthe specific binding affinity and specific binding avidity,respectively, of a protein for its counter-structure under specificbinding conditions. In biochemical kinetics avidity refers to theaccumulated strength of multiple affinities of individual non-covalentbinding interactions, such as between ICOSL and its counter-structuresICOS and/or CD28. As such, avidity is distinct from affinity, whichdescribes the strength of a single interaction. An increase orattenuation in binding affinity of a variant ICOSL containing anaffinity modified ICOSL IgSF domain to its counter-structure isdetermined relative to the binding affinity of the unmodified ICOSL,such as an unmodified ICOSL containing the native or wild-type IgSFdomain, such as IgV domain. Methods for determining binding affinity oravidity are known in art. See, for example, Larsen et al., AmericanJournal of Transplantation, Vol 5: 443-453 (2005). In some embodiments,a variant ICOSL of the invention (i.e. a ICOSL protein containing anaffinity modified IgSF domain) specifically binds to CD28 and/or ICOSmeasured by flow cytometry with a binding affinity that yields a MeanFluorescence Intensity (MFI) value at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 100% greater than a wild-type ICOSL control in abinding assay such as described in Example 6.

The term “biological half-life” refers to the amount of time it takesfor a substance, such as an immunomodulatory polypeptide comprising avariant ICOSL of the present invention, to lose half of itspharmacologic or physiologic activity or concentration. Biologicalhalf-life can be affected by elimination, excretion, degradation (e.g.,enzymatic) of the substance, or absorption and concentration in certainorgans or tissues of the body. In some embodiments, biological half-lifecan be assessed by determining the time it takes for the blood plasmaconcentration of the substance to reach half its steady state level(“plasma half-life”). Conjugates that can be used to derivatize andincrease the biological half-life of polypeptides of the invention areknown in the art and include, but are not limited to, polyethyleneglycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinantpeptides; see, WO2013130683), human serum albumin (HSA), bovine serumalbumin (BSA), lipids (acylation), and poly-Pro-Ala-Ser (PAS),polyglutamic acid (glutamylation).

The term “chimeric antigen receptor” or “CAR” as used herein refers toan artificial (i.e., man-made) transmembrane protein expressed on amammalian cell comprising at least an ectodomain, a transmembrane, andan endodomain. Optionally, the CAR protein includes a “spacer” whichcovalently links the ectodomain to the transmembrane domain. A spacer isoften a polypeptide linking the ectodomain to the transmembrane domainvia peptide bonds. The CAR is typically expressed on a mammalianlymphocyte. In some embodiments, the CAR is expressed on a mammaliancell such as a T-cell or a tumor infiltrating lymphocyte (TIL). A CARexpressed on a T-cell is referred to herein as a “CAR T-cell” or“CAR-T.” In some embodiments the CAR-T is a T helper cell, a cytotoxicT-cell, a natural killer T-cell, a memory T-cell, a regulatory T-cell,or a gamma delta T-cell. When used clinically in, e.g. adoptive celltransfer, a CAR-T with antigen binding specificity to the patient'stumor is typically engineered to express on a native T-cell obtainedfrom the patient. The engineered T-cell expressing the CAR is theninfused back into the patient. The CAR-T is thus often an autologousCAR-T although allogeneic CAR-T are included within the scope of theinvention. The ectodomain of a CAR comprises an antigen binding region,such as an antibody or antigen binding fragment thereof (e.g. scFv),that specifically binds under physiological conditions with a targetantigen, such as a tumor specific antigen. Upon specific binding abiochemical chain of events (i.e., signal transduction) results inmodulation of the immunological activity of the CAR-T. Thus, forexample, upon specific binding by the antigen binding region of theCAR-T to its target antigen can lead to changes in the immunologicalactivity of the T-cell activity as reflected by changes in cytotoxicity,proliferation or cytokine production. Signal transduction upon CAR-Tactivation is achieved in some embodiments by the CD3-zeta chain(“CD3-z”) which is involved in signal transduction in native mammalianT-cells. CAR-Ts can further comprise multiple signaling domains such asCD28, 41BB or OX40, to further modulate immunomodulatory response of theT-cell. CD3-z comprises a conserved motif known as an immunoreceptortyrosine-based activation motif (ITAM) which is involved in T-cellreceptor signal transduction.

The term “collectively” or “collective” when used in reference tocytokine production induced by the presence of two or more variant ICOSLof the invention in an in vitro assay, means the overall cytokineexpression level irrespective of the cytokine production induced byindividual variant ICOSL. In some embodiments, the cytokine beingassayed is IFN-gamma in an in vitro primary T-cell assay such asdescribed in Example 6 and Example 7.

The term “cognate binding partner” (used interchangeably with“counter-structure”) in reference to a polypeptide, such as in referenceto an IgSF domain of a variant ICOSL, refers to at least one molecule(typically a native mammalian protein) to which the referencedpolypeptide specifically binds under specific binding conditions. Insome aspects, a variant ICOSL containing an affinity modified IgSFdomain specifically binds to the counter-structure of the correspondingnative or wild-type ICOSL but with increased or attenuated affinity. Aspecies of ligand recognized and specifically binding to its cognatereceptor under specific binding conditions is an example of acounter-structure or cognate binding partner of that receptor. A“cognate cell surface binding partner” is a cognate binding partnerexpressed on a mammalian cell surface. A “cell surface molecularspecies” is a cognate binding partner of ligands of the immunologicalsynapse (IS), expressed on and by cells, such as mammalian cells,forming the immunological synapse.

As used herein, “conjugate,” “conjugation” or grammatical variationsthereof refers the joining or linking together of two or more compoundsresulting in the formation of another compound, by any joining orlinking methods known in the art. It can also refer to a compound whichis generated by the joining or linking together two or more compounds.For example, a variant ICOSL polypeptide linked directly or indirectlyto one or more chemical moieties or polypeptide is an exemplaryconjugate. Such conjugates include fusion proteins, those produced bychemical conjugates and those produced by any other methods.

The term “competitive binding” as used herein means that a protein iscapable of specifically binding to at least two cognate binding partnersbut that specific binding of one cognate binding partner inhibits, suchas prevents or precludes, simultaneous binding of the second cognatebinding partner. Thus, in some cases, it is not possible for a proteinto bind the two cognate binding partners at the same time. Generally,competitive binders contain the same or overlapping binding site forspecific binding but this is not a requirement. In some embodiments,competitive binding causes a measurable inhibition (partial or complete)of specific binding of a protein to one of its cognate binding partnerdue to specific binding of a second cognate binding partner. A varietyof methods are known to quantify competitive binding such as ELISA(enzyme linked immunosorbent assay) assays.

The term “conservative amino acid substitution” as used herein means anamino acid substitution in which an amino acid residue is substituted byanother amino acid residue having a side chain R group with similarchemical properties (e.g., charge or hydrophobicity). Examples of groupsof amino acids that have side chains with similar chemical propertiesinclude 1) aliphatic side chains: glycine, alanine, valine, leucine, andisoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3)amide-containing side chains: asparagine and glutamine; 4) aromatic sidechains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains:lysine, arginine, and histidine; 6) acidic side chains: aspartic acidand glutamic acid; and 7) sulfur-containing side chains: cysteine andmethionine. Conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.

The term, “corresponding to” with reference to positions of a protein,such as recitation that nucleotides or amino acid positions “correspondto” nucleotides or amino acid positions in a disclosed sequence, such asset forth in the Sequence listing, refers to nucleotides or amino acidpositions identified upon alignment with the disclosed sequence based onstructural sequence alignment or using a standard alignment algorithm,such as the GAP algorithm. For example, corresponding residues can bedetermined by alignment of a reference sequence with the sequence setforth in SEQ ID NO: 32 (ECD domain) or set forth in SEQ ID NOs: 196 or545 (IgV domain) by structural alignment methods as described herein. Byaligning the sequences, one skilled in the art can identifycorresponding residues, for example, using conserved and identical aminoacid residues as guides.

The terms “decrease” or “attenuate” “or suppress” as used herein meansto decrease by a statistically significant amount. A decrease can be atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of a controlvalue, such as a non-zero control value.

The terms “decreased” or “reduced” as used herein in the context ofdecreasing immunological activity of a mammalian lymphocyte means todecrease one or more activities of the lymphocyte, as compared to acontrol, such as an untreated control or a control in which a treatmentusing an unmodified or non-variant control was employed under the sameconditions. A decreased activity can refer to one or more of cell cycleinhibition, reduced cell survival, reduced cell proliferation, reducedcytokine production, or reduced T-cell cytotoxicity, such as by astatistically significant amount. In some embodiments, reference toreduced immunological activity means to reduce interferon gamma(IFN-gamma) production compared to in the absence of treatment, such asby a statistically significant amount. In some embodiments, theimmunological activity can be assessed in a mixed lymphocyte reaction(MLR) assay. Methods of conducting MLR assays are known in the art. Wanget al., Cancer Immunol Res. 2014 September: 2(9):846-56. Other methodsof assessing activities of lymphocytes are known in the art, includingany assay as described herein. In some embodiments an enhancement can bea decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,60%, 70%, 80%, 90%, or 100%, as compared to a control value, such as anuntreated control value or a non-zero control value.

The terms “derivatives” or “derivatized” refer to modification of aprotein by covalently linking it, directly or indirectly, to acomposition so as to alter such characteristics as biological half-life,bioavailability, immunogenicity, solubility, toxicity, potency, orefficacy while retaining or enhancing its therapeutic benefit.Derivatives of immunomodulatory polypeptides of the invention are withinthe scope of the invention and can be made by, for example,glycosylation, pegylation, lipidation, or Fc-fusion.

As used herein, domain (typically a sequence of three or more, generally5 or 7 or more amino acids, such as 10 to 200 amino acid residues)refers to a portion of a molecule, such as a protein or encoding nucleicacid, that is structurally and/or functionally distinct from otherportions of the molecule and is identifiable. For example, domainsinclude those portions of a polypeptide chain that can form anindependently folded structure within a protein made up of one or morestructural motifs and/or that is recognized by virtue of a functionalactivity, such as binding activity. A protein can have one, or more thanone, distinct domains. For example, a domain can be identified, definedor distinguished by homology of the primary sequence or structure torelated family members, such as homology to motifs. In another example,a domain can be distinguished by its function, such as an ability tointeract with a biomolecule, such as a cognate binding partner. A domainindependently can exhibit a biological function or activity such thatthe domain independently or fused to another molecule can perform anactivity, such as, for example binding. A domain can be a linearsequence of amino acids or a non-linear sequence of amino acids. Manypolypeptides contain a plurality of domains. Such domains are known, andcan be identified by those of skill in the art. For exemplificationherein, definitions are provided, but it is understood that it is wellwithin the skill in the art to recognize particular domains by name. Ifneeded appropriate software can be employed to identify domains.

The term “ectodomain” as used herein refers to the region of a membraneprotein, such as a transmembrane protein, that lies outside thevesicular membrane. Ectodomains often comprise binding domains thatspecifically bind to ligands or cell surface receptors, such as via abinding domain that specifically binds to the ligand or cell surfacereceptor. The ectodomain of a cellular transmembrane protein isalternately referred to as an extracellular domain.

The terms “effective amount” or “therapeutically effective amount” referto a quantity and/or concentration of a therapeutic composition of theinvention, including a protein composition or cell composition, thatwhen administered ex vivo (by contact with a cell from a patient) or invivo (by administration into a patient) either alone (i.e., as amonotherapy) or in combination with additional therapeutic agents,yields a statistically significant decrease in disease progression as,for example, by ameliorating or eliminating symptoms and/or the cause ofthe disease. An effective amount may be an amount that relieves,lessens, or alleviates at least one symptom or biological response oreffect associated with a disease or disorder, prevents progression ofthe disease or disorder, or improves physical functioning of thepatient. In the case of cell therapy, the effective amount is aneffective dose or number of cells administered to a patient by adoptivecell therapy. In some embodiments the patient is a mammal such as anon-human primate or human patient.

The term “endodomain” as used herein refers to the region found in somemembrane proteins, such as transmembrane proteins, that extends into theinterior space defined by the cell surface membrane. In mammalian cells,the endodomain is the cytoplasmic region of the membrane protein. Incells, the endodomain interacts with intracellular constituents and canbe play a role in signal transduction and thus, in some cases, can be anintracellular signaling domain. The endodomain of a cellulartransmembrane protein is alternately referred to as a cytoplasmicdomain, which, in some cases, can be a cytoplasmic signaling domain.

The terms “enhanced” or “increased” as used herein in the context ofincreasing immunological activity of a mammalian lymphocyte means toincrease one or more activities the lymphocyte, as compared to acontrol, such as an untreated control or a control in which a treatmentusing an unmodified or non-variant control was employed under the sameconditions. An increased activity can be one or more of increase cellsurvival, cell proliferation, cytokine production, or T-cellcytotoxicity, such as by a statistically significant amount. In someembodiments, reference to increased immunological activity means toincrease interferon gamma (IFN-gamma) production, such as by astatistically significant amount. In some embodiments, the immunologicalactivity can be assessed in a mixed lymphocyte reaction (MLR) assay.Methods of conducting MLR assays are known in the art. Wang et al.,Cancer Immunol Res. 2014 September: 2(9):846-56. Other methods ofassessing activities of lymphocytes are known in the art, including anyassay as described herein. In some embodiments an enhancement can be anincrease of at least 10%, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%,400%, or 500% greater than a non-zero control value.

The term “engineered cell” as used herein refers to a mammalian cellthat has been genetically modified by human intervention such as byrecombinant DNA methods or viral transduction. In some embodiments, thecell is an immune cell, such as a lymphocyte (e.g. T cell, B cell, NKcell) or an antigen presenting cell (e.g. dendritic cell). The cell canbe a primary cell from a patient or can be a cell line. In someembodiments, an engineered cell of the invention comprises a variantICOSL provided herein. In some embodiments, the variant ICOSL is atransmembrane immunomodulatory protein (hereinafter referred to as“TIP”) that is expressed on the engineered cell. In some embodiments,the TIP contains the extracellular domain or a portion thereofcontaining the IgV domain linked to a transmembrane domain (e.g., aICOSL transmembrane domain) and, optionally, an intracellular signalingdomain. In some cases, the TIP is formatted as a chimeric receptorcontaining a heterologous cytoplasmic signaling domain or endodomain. Insome embodiments, an engineered cell is capable of expressing andsecreting a immunomodulatory protein as described herein. Among providedengineered cells also are cells further containing an engineered T-cellreceptor (TCR) or chimeric antigen receptor (CAR).

The term “engineered T-cell” as used herein refers to a T-cell such as aT helper cell, cytotoxic T-cell (alternatively, cytotoxic T lymphocyteor CTL), natural killer T-cell, regulatory T-cell, memory T-cell, orgamma delta T-cell, that has been genetically modified by humanintervention such as by recombinant DNA methods or viral transductionmethods. An engineered T-cell comprises a variant ICOSL transmembraneimmunomodulatory protein (TIP) or secreted immunodulatory protein (SIP)of the present invention that is expressed on the T-cell and isengineered to modulate immunological activity of the engineered T-cellitself, or a mammalian cell to which the variant ICOSL expressed on theT-cell specifically binds. An engineered T-cell can comprise a variantICOSL secreted immunomodulatory protein (SIP) of the present inventionthat is expressed by and/or secreted by the T-cell and is engineered tomodulate immunological activity of the engineered T-cell itself, or amammalian cell to which the variant ICOSL when secreted by the T-cell,specifically binds.

The term “engineered T-cell receptor” or “engineered TCR” refers to aT-cell receptor (TCR) engineered to specifically bind with a desiredaffinity to a major histocompatibility complex (MHC)/peptide targetantigen that is selected, cloned, and/or subsequently introduced into apopulation of T-cells, often used for adoptive immunotherapy. Incontrast to engineered TCRs, CARs are engineered to bind target antigensin a MHC independent manner.

The term “expressed on” as used herein is used in reference to a proteinexpressed on the surface of a cell, such as a mammalian cell. Thus, theprotein is expressed as a membrane protein. In some embodiments, theexpressed protein is a transmembrane protein. In some embodiments, theprotein is conjugated to a small molecule moiety such as a drug ordetectable label. Proteins expressed on the surface of a cell caninclude cell-surface proteins such as cell surface receptors that areexpressed on mammalian cells.

The term “half-life extending moiety” refers to a moiety of apolypeptide fusion or chemical conjugate that extends the half-life of aprotein circulating in mammalian blood serum compared to the half-lifeof the protein that is not so conjugated to the moiety. In someembodiments, half-life is extended by greater than or greater than about1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, or 6.0-fold.In some embodiments, half-life is extended by more than 6 hours, morethan 12 hours, more than 24 hours, more than 48 hours, more than 72hours, more than 96 hours or more than 1 week after in vivoadministration compared to the protein without the half-life extendingmoiety. The half-life refers to the amount of time it takes for theprotein to lose half of its concentration, amount, or activity.Half-life can be determined for example, by using an ELISA assay or anactivity assay. Exemplary half-life extending moieties include an Fcdomain, a multimerization domain, polyethylene glycol (PEG),hydroxyethyl starch (HES), XTEN (extended recombinant peptides; see,WO2013130683), human serum albumin (HSA), bovine serum albumin (BSA),lipids (acylation), and poly-Pro-Ala-Ser (PAS), and polyglutamic acid(glutamylation).

The term “immunological synapse” or “immune synapse” as used hereinmeans the interface between a mammalian cell that expresses MHC I (majorhistocompatibility complex) or MHC II, such as an antigen-presentingcell or tumor cell, and a mammalian lymphocyte such as an effector Tcell or Natural Killer (NK) cell.

An Fc (fragment crystallizable) region or domain of an immunoglobulinmolecule (also termed an Fc polypeptide) corresponds largely to theconstant region of the immunoglobulin heavy chain, and is responsiblefor various functions, including the antibody's effector function(s).The Fc domain contains part or all of a hinge domain of animmunoglobulin molecule plus a CH2 and a CH3 domain. The Fc domain canform a dimer of two polypeptide chains joined by one or more disulfidebonds. In some embodiments, the Fc is a variant Fc that exhibits reduced(e.g. reduced greater than 30%, 40%, 50%, 60%, 70%, 80%, 90% or more)activity to facilitate an effector function. In some embodiments,reference to amino acid substitutions in an Fc region is by EU numberingsystem unless described with reference to a specific SEQ ID NO. EUnumbering is known and is according to the most recently updated IMGTScientific Chart (IMGT®, the international ImMunoGeneTics informationSystem®http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html(created: 17 May 2001, last updated: 10 Jan. 2013) and the EU index asreported in Kabat, E. A. et al. Sequences of Proteins of Immunologicalinterest. 5th ed. US Department of Health and Human Services, NIHpublication No. 91-3242 (1991).

An immunoglobulin Fc fusion (“Fc-fusion”), such as an immunomodulatoryFc fusion protein, is a molecule comprising one or more polypeptides (orone or more small molecules) operably linked to an Fc region of animmunoglobulin. An Fc-fusion may comprise, for example, the Fc region ofan antibody (which facilitates effector functions and pharmacokinetics)and a variant ICOSL. An immunoglobulin Fc region may be linkedindirectly or directly to one or more variant ICOSL or small molecules(fusion partners). Various linkers are known in the art and canoptionally be used to link an Fc to a fusion partner to generate anFc-fusion. Fc-fusions of identical species can be dimerized to formFc-fusion homodimers, or using non-identical species to form Fc-fusionheterodimers. In some embodiments, the Fc is a mammalian Fc such as amurine or human Fc.

The term “host cell” refers to a cell that can be used to express aprotein encoded by a recombinant expression vector. A host cell can be aprokaryote, for example, E. coli, or it can be a eukaryote, for example,a single-celled eukaryote (e.g., a yeast or other fungus), a plant cell(e.g., a tobacco or tomato plant cell), an animal cell (e.g., a humancell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or aninsect cell) or a hybridoma. Examples of host cells include Chinesehamster ovary (CHO) cells or their derivatives such as Veggie CHO andrelated cell lines which grow in serum-free media or CHO strain DX-B11,which is deficient in DHFR. In some embodiments, a host cell is amammalian cell (e.g., a human cell, a monkey cell, a hamster cell, a ratcell, a mouse cell, or an insect cell).

The term “immunoglobulin” (abbreviated “Ig”) as used herein refers to amammalian immunoglobulin protein including any of the five human classesof antibody: IgA (which includes subclasses IgA1 and IgA2), IgD, IgE,IgG (which includes subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. Theterm is also inclusive of immunoglobulins that are less thanfull-length, whether wholly or partially synthetic (e.g., recombinant orchemical synthesis) or naturally produced, such as antigen bindingfragment (Fab), variable fragment (Fv) containing V_(H) and V_(L), thesingle chain variable fragment (scFv) containing V_(H) and V_(L) linkedtogether in one chain, as well as other antibody V region fragments,such as Fab′, F(ab)₂, F(ab′)₂, dsFv diabody, Fc, and Fd polypeptidefragments. Bispecific antibodies, homobispecific and heterobispecific,are included within the meaning of the term.

The term “immunoglobulin superfamily” or “IgSF” as used herein means thegroup of cell surface and soluble proteins that are involved in therecognition, binding, or adhesion processes of cells. Molecules arecategorized as members of this superfamily based on shared structuralfeatures with immunoglobulins (i.e., antibodies); they all possess adomain known as an immunoglobulin domain or fold. Members of the IgSFinclude cell surface antigen receptors, co-receptors and co-stimulatorymolecules of the immune system, molecules involved in antigenpresentation to lymphocytes, cell adhesion molecules, certain cytokinereceptors and intracellular muscle proteins. They are commonlyassociated with roles in the immune system. Proteins in theimmunological synapse are often members of the IgSF. IgSF can also beclassified into “subfamilies” based on shared properties such asfunction. Such subfamilies typically consist of from 4 to 30 IgSFmembers.

The terms “IgSF domain” or “immunoglobulin domain” or “Ig domain” asused herein refers to a structural domain of IgSF proteins. Ig domainsare named after the immunoglobulin molecules. They contain about 70-110amino acids and are categorized according to their size and function.Ig-domains possess a characteristic Ig-fold, which has a sandwich-likestructure formed by two sheets of antiparallel beta strands.Interactions between hydrophobic amino acids on the inner side of thesandwich and highly conserved disulfide bonds formed between cysteineresidues in the B and F strands, stabilize the Ig-fold. One end of theIg domain has a section called the complementarity determining regionthat is important for the specificity of antibodies for their ligands.The Ig like domains can be classified (into classes) as: IgV, IgC1,IgC2, or IgI. Most Ig domains are either variable (IgV) or constant(IgC). IgV domains with 9 beta strands are generally longer than IgCdomains with 7 beta strands. Ig domains of some members of the IgSFresemble IgV domains in the amino acid sequence, yet are similar in sizeto IgC domains. These are called IgC2 domains, while standard IgCdomains are called IgC1 domains. T-cell receptor (TCR) chains containtwo Ig domains in the extracellular portion; one IgV domain at theN-terminus and one IgC1 domain adjacent to the cell membrane. ICOSLcontains two Ig domains: IgV and IgC.

The term “IgSF species” as used herein means an ensemble of IgSF memberproteins with identical or substantially identical primary amino acidsequence. Each mammalian immunoglobulin superfamily (IgSF) memberdefines a unique identity of all IgSF species that belong to that IgSFmember. Thus, each IgSF family member is unique from other IgSF familymembers and, accordingly, each species of a particular IgSF familymember is unique from the species of another IgSF family member.Nevertheless, variation between molecules that are of the same IgSFspecies may occur owing to differences in post-translationalmodification such as glycosylation, phosphorylation, ubiquitination,nitrosylation, methylation, acetylation, and lipidation. Additionally,minor sequence differences within a single IgSF species owing to genepolymorphisms constitute another form of variation within a single IgSFspecies as do wild type truncated forms of IgSF species owing to, forexample, proteolytic cleavage. A “cell surface IgSF species” is an IgSFspecies expressed on the surface of a cell, generally a mammalian cell.

The term “immunological activity” as used herein in the context ofmammalian lymphocytes such as T-cells refers to one or more cellsurvival, cell proliferation, cytokine production (e.g.interferon-gamma), or T-cell cytotoxicity activities. In some cases, animmunological activity can mean the cell expression of cytokines, suchas chemokines or interleukins. Assays for determining enhancement orsuppression of immunological activity include the MLR (mixed lymphocytereaction) assays measuring interferon-gamma cytokine levels in culturesupernatants (Wang et al., Cancer Immunol Res. 2014 September:2(9):846-56), SEB (staphylococcal enterotoxin B) T cell stimulationassay (Wang et al., Cancer Immunol Res. 2014 September: 2(9):846-56),and anti-CD3 T cell stimulation assays (Li and Kurlander, J Transl Med.2010: 8: 104). Since T cell activation is associated with secretion ofIFN-gamma cytokine, detecting IFN-gamma levels in culture supernatantsfrom these in vitro human T cell assays can be assayed using commercialELISA kits (Wu et al, Immunol Lett 2008 Apr. 15; 117(1): 57-62).Induction of an immune response results in an increase in immunologicalactivity relative to quiescent lymphocytes. An immunomodulatory protein,such as a variant ICOSL polypeptide containing an affinity modified IgSFdomain, as provided herein can in some embodiments increase or, inalternative embodiments, decrease IFN-gamma (interferon-gamma)expression in a primary T-cell assay relative to a wild-type IgSF memberor IgSF domain control. Those of skill will recognize that the format ofthe primary T-cell assay used to determine an increase in IFN-gammaexpression will differ from that employed to assay for a decrease inIFN-gamma expression. In assaying for the ability of an immunomodulatoryprotein or affinity modified IgSF domain of the invention to decreaseIFN-gamma expression in a primary T-cell assay, a Mixed LymphocyteReaction (MLR) assay can be used as described in Example 6.Conveniently, a soluble form of an affinity modified IgSF domain of theinvention can be employed to determine its ability to antagonize andthereby decrease the IFN-gamma expression in a MLR as likewise describedin Example 6. Alternatively, in assaying for the ability of animmunomodulatory protein or affinity modified IgSF domain of theinvention to increase IFN-gamma expression in a primary T-cell assay, aco-immobilization assay can be used. In a co-immobilization assay, aT-cell receptor signal, provided in some embodiments by anti-CD3antibody, is used in conjunction with a co-immobilized affinity modifiedIgSF domain, such as variant ICOSL, to determine the ability to increaseIFN-gamma expression relative to a wild-type IgSF domain control.Methods to assay the immunological activity of engineered cells,including to evaluate the activity of a variant ICOSL transmembraneimmunomodulatory protein, are known in the art and include, but are notlimited to, the ability to expand T cells following antigen stimulation,sustain T cell expansion in the absence of re-stimulation, andanti-cancer activities in appropriate animal models. Assays also includeassays to assess cytotoxicity, including a standard ⁵¹Cr-release assay(see e.g. Milone et al., (2009) Molecular Therapy 17: 1453-1464) or flowbased cytotoxicity assays, or an impedance based cytotoxicity assay(Peper et al. (2014) Journal of Immunological Methods, 405:192-198).

An “immunomodulatory polypeptide” or “immunomodulatory protein” is apolypeptide or protein molecule that modulates immunological activity.By “modulation” or “modulating” an immune response is meant thatimmunological activity is either increased or decreased. Animmunomodulatory protein can be a single polypeptide chain or a multimer(dimers or higher order multimers) of at least two polypeptide chainscovalently bonded to each other by, for example, interchain disulfidebonds. Thus, monomeric, dimeric, and higher order multimericpolypeptides are within the scope of the defined term. Multimericpolypeptides can be homomultimeric (of identical polypeptide chains) orheteromultimeric (of non-identical polypeptide chains). Animmunomodulatory protein of the invention comprises a variant ICOSL.

The term “increase” as used herein means to increase by a statisticallysignificant amount. An increase can be at least 5%, 10%, 20%, 30%, 40%,50%, 75%, 100%, or greater than a non-zero control value.

An “isoform” of ICOSL (inducible costimulator ligand; CD275) is one of aplurality of naturally occurring ICOSL polypeptides that differ in aminoacid sequence. Isoforms can be the product of splice variants of an RNAtranscript expressed by a single gene, or the expression product ofhighly similar but different genes yielding a functionally similarprotein such as may occur from gene duplication. As used herein, theterm “isoform” of ICOSL also refers to the product of different allelesof an ICOSL gene (e.g., ICOSLG).

The term “lymphocyte” as used herein means any of three subtypes ofwhite blood cell in a mammalian immune system. They include naturalkiller cells (NK cells) (which function in cell-mediated, cytotoxicinnate immunity), T cells (for cell-mediated, cytotoxic adaptiveimmunity), and B cells (for humoral, antibody-driven adaptive immunity).T cells include: T helper cells, cytotoxic T-cells, natural killerT-cells, memory T-cells, regulatory T-cells, or gamma delta T-cells.Innate lymphoid cells (ILC) are also included within the definition oflymphocyte.

The terms “mammal,” or “patient” specifically includes reference to atleast one of a: human, chimpanzee, rhesus monkey, cynomolgus monkey,dog, cat, mouse, or rat.

The term “membrane protein” as used herein means a protein that, underphysiological conditions, is attached directly or indirectly to a lipidbilayer. A lipid bilayer that forms a membrane can be a biologicalmembrane such as a eukaryotic (e.g., mammalian) cell membrane or anartificial (i.e., man-made) membrane such as that found on a liposome.Attachment of a membrane protein to the lipid bilayer can be by way ofcovalent attachment, or by way of non-covalent interactions such ashydrophobic or electrostatic interactions. A membrane protein can be anintegral membrane protein or a peripheral membrane protein. Membraneproteins that are peripheral membrane proteins are non-covalentlyattached to the lipid bilayer or non-covalently attached to an integralmembrane protein. A peripheral membrane protein forms a temporaryattachment to the lipid bilayer such that under the range of conditionsthat are physiological in a mammal, peripheral membrane protein canassociate and/or disassociate from the lipid bilayer. In contrast toperipheral membrane proteins, integral membrane proteins form asubstantially permanent attachment to the membrane's lipid bilayer suchthat under the range of conditions that are physiological in a mammal,integral membrane proteins do not disassociate from their attachment tothe lipid bilayer. A membrane protein can form an attachment to themembrane by way of one layer of the lipid bilayer (monotopic), orattached by way of both layers of the membrane (polytopic). An integralmembrane protein that interacts with only one lipid bilayer is an“integral monotopic protein”. An integral membrane protein thatinteracts with both lipid bilayers is an “integral polytopic protein”alternatively referred to herein as a “transmembrane protein”.

The terms “modulating” or “modulate” as used herein in the context of animmune response, such as a mammalian immune response, refer to anyalteration, such as an increase or a decrease, of existing or potentialimmune responses that occurs as a result of administration of animmunomodulatory polypeptide comprising a variant ICOSL of the presentinvention or as a result of administration of engineered cells expressesan immunomodulatory protein, such as a variant ICOSL transmembraneimmunomodulatory protein of the present invention. Thus, it refers to analteration, such as an increase or decrease, of an immune response ascompared to the immune response that occurs or is present in the absenceof the administration of the immunomodulatory protein comprising thevariant ICOSL or cells expressing such an immunomodulatory polypeptide.Such modulation includes any induction, activation, suppression oralteration in degree or extent of immunological activity of an immunecell. Immune cells include B cells, T cells, NK (natural killer) cells,NK T cells, professional antigen-presenting cells (APCs), andnon-professional antigen-presenting cells, and inflammatory cells(neutrophils, macrophages, monocytes, eosinophils, and basophils).Modulation includes any change imparted on an existing immune response,a developing immune response, a potential immune response, or thecapacity to induce, regulate, influence, or respond to an immuneresponse. Modulation includes any alteration in the expression and/orfunction of genes, proteins and/or other molecules in immune cells aspart of an immune response. Modulation of an immune response ormodulation of immunological activity includes, for example, thefollowing: elimination, deletion, or sequestration of immune cells;induction or generation of immune cells that can modulate the functionalcapacity of other cells such as autoreactive lymphocytes, antigenpresenting cells, or inflammatory cells; induction of an unresponsivestate in immune cells (i.e., anergy); enhancing or suppressing theactivity or function of immune cells, including but not limited toaltering the pattern of proteins expressed by these cells. Examplesinclude altered production and/or secretion of certain classes ofmolecules such as cytokines, chemokines, growth factors, transcriptionfactors, kinases, costimulatory molecules, or other cell surfacereceptors or any combination of these modulatory events. Modulation canbe assessed, for example, by an alteration in IFN-gamma (interferongamma) expression relative to the wild-type ICOSL control in a primary Tcell assay (see, Zhao and Ji, Exp Cell Res. 2016 Jan. 1; 340(1)132-138). Modulation can be assessed, for example, by an alteration ofan immunological activity of engineered cells, such as an alteration incytotoxic activity of engineered cells or an alteration in cytokinesecretion of engineered cells relative to cells engineered with awild-type ICOSL transmembrane protein.

The term “molecular species” as used herein means an ensemble ofproteins with identical or substantially identical primary amino acidsequence. Each mammalian immunoglobulin superfamily (IgSF) memberdefines a collection of identical or substantially identical molecularspecies. Thus, for example, human ICOSL is an IgSF member and each humanICOSL molecule is a molecule species of ICOS. Variation betweenmolecules that are of the same molecular species may occur owing todifferences in post-translational modification such as glycosylation,phosphorylation, ubiquitination, nitrosylation, methylation,acetylation, and lipidation. Additionally, minor sequence differenceswithin a single molecular species owing to gene polymorphisms constituteanother form of variation within a single molecular species as do wildtype truncated forms of a single molecular species owing to, forexample, proteolytic cleavage. A “cell surface molecular species” is amolecular species expressed on the surface of a mammalian cell. Two ormore different species of protein, each of which is present exclusivelyon one or exclusively the other (but not both) of the two mammaliancells forming the IS, are said to be in “cis” or “cis configuration”with each other. Two different species of protein, the first of which isexclusively present on one of the two mammalian cells forming the IS andthe second of which is present exclusively on the second of the twomammalian cells forming the IS, are said to be in “trans” or “transconfiguration.” Two different species of protein each of which ispresent on both of the two mammalian cells forming the IS are in bothcis and trans configurations on these cells.

The term, a “multimerization domain” refers to a sequence of amino acidsthat promotes stable interaction of a polypeptide molecule with one ormore additional polypeptide molecules, each containing a complementarymultimerization domain (e.g. a first multimerization domain and a secondmultimerization domain), which can be the same or a differentmultimerization domain. The interactions between complementarymultimerization domains, e.g. interaction between a firstmultimerization domain and a second multimerization domain, form astable protein-protein interaction to produce a multimer of thepolypeptide molecule with the additional polypeptide molecule. In somecases, the multimerization domain is the same and interacts with itselfto form a stable protein-protein interaction between two polypeptidechains. Generally, a polypeptide is joined directly or indirectly to themultimerization domain. Exemplary multimerization domains include theimmunoglobulin sequences or portions thereof, leucine zippers,hydrophobic regions, hydrophilic regions, and compatible protein-proteininteraction domains. The multimerization domain, for example, can be animmunoglobulin constant region or domain, such as, for example, the Fcdomain or portions thereof from IgG, including IgG1, IgG2, IgG3 or IgG4subtypes, IgA, IgE, IgD and IgM and modified forms thereof.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyto refer to a polymer of nucleic acid residues (e.g.,deoxyribonucleotides or ribonucleotides) in either single- ordouble-stranded form. Unless specifically limited, the terms encompassnucleic acids containing known analogues of natural nucleotides and thathave similar binding properties to it and are metabolized in a mannersimilar to naturally-occurring nucleotides. Unless otherwise indicated,a particular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary nucleotide sequences as well as thesequence explicitly indicated (a “reference sequence”). Specifically,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues. The termnucleic acid or polynucleotide encompasses cDNA or mRNA encoded by agene.

The term “non-competitive binding” as used herein means the ability of aprotein to specifically bind simultaneously to at least two cognatebinding partners. Thus, the protein is able to bind to at least twodifferent cognate binding partners at the same time, although thebinding interaction need not be for the same duration such that, in somecases, the protein is specifically bound to only one of the cognatebinding partners. In some embodiments, the binding occurs under specificbinding conditions. In some embodiments, the simultaneous binding issuch that binding of one cognate binding partner does not substantiallyinhibit simultaneous binding to a second cognate binding partner. Insome embodiments, non-competitive binding means that binding a secondcognate binding partner to its binding site on the protein does notdisplace the binding of a first cognate binding partner to its bindingsite on the protein. Methods of assessing non-competitive binding arewell known in the art such as the method described in Perez de La Lastraet al., Immunology, 1999 April: 96(4): 663-670. In some cases, innon-competitive interactions, the first cognate binding partnerspecifically binds at an interaction site that does not overlap with theinteraction site of the second cognate binding partner such that bindingof the second cognate binding partner does not directly interfere withthe binding of the first cognate binding partner. Thus, any effect onbinding of the cognate binding partner by the binding of the secondcognate binding partner is through a mechanism other than directinterference with the binding of the first cognate binding partner. Forexample, in the context of enzyme-substrate interactions, anon-competitive inhibitor binds to a site other than the active site ofthe enzyme. Non-competitive binding encompasses uncompetitive bindinginteractions in which a second cognate binding partner specificallybinds at an interaction site that does not overlap with the binding ofthe first cognate binding partner but binds to the second interactionsite only when the first interaction site is occupied by the firstcognate binding partner.

The term “pharmaceutical composition” refers to a composition suitablefor pharmaceutical use in a mammalian subject, often a human. Apharmaceutical composition typically comprises an effective amount of anactive agent (e.g., an immunomodulatory polypeptide comprising a variantICOSL or engineered cells expressing a variant ICOSL transmembraneimmunomodulatory protein) and a carrier, excipient, or diluent. Thecarrier, excipient, or diluent is typically a pharmaceuticallyacceptable carrier, excipient or diluent, respectively.

The terms “polypeptide” and “protein” are used interchangeably hereinand refer to a molecular chain of two or more amino acids linked throughpeptide bonds. The terms do not refer to a specific length of theproduct. Thus, “peptides,” and “oligopeptides,” are included within thedefinition of polypeptide. The terms include post-translationalmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like. The terms also includemolecules in which one or more amino acid analogs or non-canonical orunnatural amino acids are included as can be synthesized, or expressedrecombinantly using known protein engineering techniques. In addition,proteins can be derivatized.

The term “primary T-cell assay” as used herein refers to an in vitroassay to measure interferon-gamma (“IFN-gamma”) expression. A variety ofsuch primary T-cell assays are known in the art such as that describedin Example 7. In a preferred embodiment, the assay used is an anti-CD3coimmobilizaton assay. In this assay, primary T cells are stimulated byanti-CD3 immobilized with or without additional recombinant proteins.Culture supernatants are harvested at timepoints, usually 24-72 hours.In another embodiment, the assay used is the MLR. In this assay, primaryT cells are stimulated with allogeneic APC. Culture supernatants areharvested at timepoints, usually 24-72 hours. Human IFN-gamma levels aremeasured in culture supernatants by standard ELISA techniques.Commercial kits are available from vendors and the assay is performedaccording to manufacturer's recommendation.

The term “purified” as applied to nucleic acids, such as encodingimmunomodulatory proteins of the invention, generally denotes a nucleicacid or polypeptide that is substantially free from other components asdetermined by analytical techniques well known in the art (e.g., apurified polypeptide or polynucleotide forms a discrete band in anelectrophoretic gel, chromatographic eluate, and/or a media subjected todensity gradient centrifugation). For example, a nucleic acid orpolypeptide that gives rise to essentially one band in anelectrophoretic gel is “purified.” A purified nucleic acid or protein ofthe invention is at least about 50% pure, usually at least about 75%,80%, 85%, 90%, 95%, 96%, 99% or more pure (e.g., percent by weight or ona molar basis).

The term “recombinant” indicates that the material (e.g., a nucleic acidor a polypeptide) has been artificially (i.e., non-naturally) altered byhuman intervention. The alteration can be performed on the materialwithin, or removed from, its natural environment or state. For example,a “recombinant nucleic acid” is one that is made by recombining nucleicacids, e.g., during cloning, affinity modification, DNA shuffling orother well-known molecular biological procedures. A “recombinant DNAmolecule,” is comprised of segments of DNA joined together by means ofsuch molecular biological techniques. The term “recombinant protein” or“recombinant polypeptide” as used herein refers to a protein moleculewhich is expressed using a recombinant DNA molecule. A “recombinant hostcell” is a cell that contains and/or expresses a recombinant nucleicacid or that is otherwise altered by genetic engineering, such as byintroducing into the cell a nucleic acid molecule encoding a recombinantprotein, such as a transmembrane immunomodulatory protein providedherein. Transcriptional control signals in eukaryotes comprise“promoter” and “enhancer” elements. Promoters and enhancers consist ofshort arrays of DNA sequences that interact specifically with cellularproteins involved in transcription. Promoter and enhancer elements havebeen isolated from a variety of eukaryotic sources including genes inyeast, insect and mammalian cells and viruses (analogous controlelements, i.e., promoters, are also found in prokaryotes). The selectionof a particular promoter and enhancer depends on what cell type is to beused to express the protein of interest. The terms “in operablecombination,” “in operable order” and “operably linked” as used hereinrefer to the linkage of nucleic acid sequences in such a manner ororientation that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced.

The term “recombinant expression vector” as used herein refers to a DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host cell. Nucleic acid sequencesnecessary for expression in prokaryotes include a promoter, optionallyan operator sequence, a ribosome binding site and possibly othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals. A secretory signal peptidesequence can also, optionally, be encoded by the recombinant expressionvector, operably linked to the coding sequence for the recombinantprotein, such as a recombinant fusion protein, so that the expressedfusion protein can be secreted by the recombinant host cell, for easierisolation of the fusion protein from the cell, if desired. The termincludes the vector as a self-replicating nucleic acid structure as wellas the vector incorporated into the genome of a host cell into which ithas been introduced. Among the vectors are viral vectors, such aslentiviral vectors.

The term “selectivity” refers to the preference of a subject protein, orpolypeptide, for specific binding of one substrate, such as one cognatebinding partner, compared to specific binding for another substrate,such as a different cognate binding partner of the subject protein.Selectivity can be reflected as a ratio of the binding activity (e.g.binding affinity) of a subject protein and a first substrate, such as afirst cognate binding partner, (e.g., K_(d1)) and the binding activity(e.g. binding affinity) of the same subject protein with a secondcognate binding partner (e.g., K_(d2)).

The term “sequence identity” as used herein refers to the sequenceidentity between genes or proteins at the nucleotide or amino acidlevel, respectively. “Sequence identity” is a measure of identitybetween proteins at the amino acid level and a measure of identitybetween nucleic acids at nucleotide level. The protein sequence identitymay be determined by comparing the amino acid sequence in a givenposition in each sequence when the sequences are aligned. Similarly, thenucleic acid sequence identity may be determined by comparing thenucleotide sequence in a given position in each sequence when thesequences are aligned. Methods for the alignment of sequences forcomparison are well known in the art, such methods include GAP, BESTFIT,BLAST, FASTA and TFASTA. The BLAST algorithm calculates percent sequenceidentity and performs a statistical analysis of the similarity betweenthe two sequences. The software for performing BLAST analysis ispublicly available through the National Center for BiotechnologyInformation (NCBI) website.

The term “soluble” as used herein in reference to proteins, means thatthe protein is not a membrane protein. In general, a soluble proteincontains only the extracellular domain of an IgSF family memberreceptor, or a portion thereof containing an IgSF domain or domains orspecific-binding fragments thereof, but does not contain thetransmembrane domain and/or is not capable of being expressed on thesurface of a cell. In some cases, solubility of a protein can beimproved by linkage or attachment, directly or indirectly via a linker,to an Fc domain, which, in some cases, also can improve the stabilityand/or half-life of the protein. In some aspects, a soluble protein isan Fc fusion protein.

The term “species” as used herein with respect to polypeptides ornucleic acids means an ensemble of molecules with identical orsubstantially identical sequences. Variation between polypeptides thatare of the same species may occur owing to differences inpost-translational modification such as glycosylation, phosphorylation,ubiquitination, nitrosylation, methylation, acetylation, and lipidation.Slightly truncated sequences of polypeptides that differ (or encode adifference) from the full length species at the amino-terminus orcarboxy-terminus by no more than 1, 2, or 3 amino acid residues areconsidered to be of a single species. Such microheterogeneities are acommon feature of manufactured proteins.

The term “specific binding fragment” as used herein in reference to afull-length wild-type mammalian ICOSL polypeptide or an IgV or an IgCdomain thereof, means a polypeptide having a subsequence of an IgVand/or IgC domain and that specifically binds in vitro and/or in vivo toa mammalian ICOS and/or mammalian CD28 such as a human or murine ICOS orCD28. In some embodiments, the specific binding fragment of ICOSL IgV orICOSL IgC is at at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% the sequence length of the full-length wild-type sequence.The specific binding fragment can be altered in sequence to form avariant ICOSL of the invention.

The term “specifically binds” as used herein means the ability of aprotein, under specific binding conditions, to bind to a target proteinsuch that its affinity or avidity is at least 5 times as great, butoptionally at least 10, 20, 30, 40, 50, 100, 250 or 500 times as great,or even at least 1000 times as great as the average affinity or avidityof the same protein to a collection of random peptides or polypeptidesof sufficient statistical size. A specifically binding protein need notbind exclusively to a single target molecule but may specifically bindto a non-target molecule due to similarity in structural conformationbetween the target and non-target (e.g., paralogs or orthologs). Thoseof skill will recognize that specific binding to a molecule having thesame function in a different species of animal (i.e., ortholog) or to anon-target molecule having a substantially similar epitope as the targetmolecule (e.g., paralog) is possible and does not detract from thespecificity of binding which is determined relative to a statisticallyvalid collection of unique non-targets (e.g., random polypeptides).Thus, a polypeptide of the invention may specifically bind to more thanone distinct species of target molecule due to cross-reactivity.Solid-phase ELISA immunoassays or Biacore measurements can be used todetermine specific binding between two proteins. Generally, interactionsbetween two binding proteins have dissociation constants (K_(d)) lessthan 1×10⁻⁵ M, and often as low as 1×10⁻¹² M. In certain embodiments ofthe present disclosure, interactions between two binding proteins havedissociation constants of 1×10⁻⁶ M, 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M,1×10⁻¹⁰ M or 1×10⁻¹¹ M.

The terms “surface expresses”, “surface expression” or “expressed on thesurface” in reference to a mammalian cell expressing a polypeptide meansthat the polypeptide is expressed as a membrane protein. In someembodiments, the membrane protein is a transmembrane protein.

As used herein, “synthetic,” with reference to, for example, a syntheticnucleic acid molecule or a synthetic gene or a synthetic peptide refersto a nucleic acid molecule or polypeptide molecule that is produced byrecombinant methods and/or by chemical synthesis methods.

The term “targeting moiety” as used herein refers to a composition thatis covalently or non-covalently attached to, or physically encapsulates,a polypeptide comprising a variant ICOSL of the present invention. Insome embodiments, the targeting moiety has specific binding affinity fora target molecule such as a target molecule expressed on a cell.Typically, the target molecule is localized on a specific tissue orcell-type. Targeting moieties include: antibodies, antigen bindingfragment (Fab), variable fragment (Fv) containing V_(H) and V_(L), thesingle chain variable fragment (scFv) containing V_(H) and V_(L) linkedtogether in one chain, as well as other antibody V region fragments,such as Fab′, F(ab)₂, F(ab′)₂, dsFv diabody, nanobodies, solublereceptors, receptor ligands, affinity matured receptors or ligands, aswell as small molecule (<500 dalton) compositions (e.g., specificbinding receptor compositions). Targeting moieties can also be attachedcovalently or non-covalently to the lipid membrane of liposomes thatencapsulate a polypeptide of the present invention.

The term “transmembrane protein” as used herein means a membrane proteinthat substantially or completely spans a lipid bilayer such as thoselipid bilayers found in a biological membrane such as a mammalian cell,or in an artificial construct such as a liposome. The transmembraneprotein comprises a transmembrane domain (“transmembrane domain”) bywhich it is integrated into the lipid bilayer and by which theintegration is thermodynamically stable under physiological conditions.Transmembrane domains are generally predictable from their amino acidsequence via any number of commercially available bioinformaticssoftware applications on the basis of their elevated hydrophobicityrelative to regions of the protein that interact with aqueousenvironments (e.g., cytosol, extracellular fluid). A transmembranedomain is often a hydrophobic alpha helix that spans the membrane. Atransmembrane protein can pass through the both layers of the lipidbilayer once or multiple times. A transmembrane protein includes theprovided transmembrane immunomodulatory proteins described herein. Inaddition to the transmembrane domain, a transmembrane immunomodulatoryprotein of the invention further comprises an ectodomain and, in someembodiments, an endodomain.

The terms “treating,” “treatment,” or “therapy” of a disease or disorderas used herein mean slowing, stopping or reversing the disease ordisorders progression, as evidenced by decreasing, cessation orelimination of either clinical or diagnostic symptoms, by administrationof a therapeutic composition (e.g. containing an immunomodulatoryprotein or engineered cells) of the invention either alone or incombination with another compound as described herein. “Treating,”“treatment,” or “therapy” also means a decrease in the severity ofsymptoms in an acute or chronic disease or disorder or a decrease in therelapse rate as for example in the case of a relapsing or remittingautoimmune disease course or a decrease in inflammation in the case ofan inflammatory aspect of an autoimmune disease. As used herein in thecontext of cancer, the terms “treatment” or, “inhibit,” “inhibiting” or“inhibition” of cancer refers to at least one of: a statisticallysignificant decrease in the rate of tumor growth, a cessation of tumorgrowth, or a reduction in the size, mass, metabolic activity, or volumeof the tumor, as measured by standard criteria such as, but not limitedto, the Response Evaluation Criteria for Solid Tumors (RECIST), or astatistically significant increase in progression free survival (PFS) oroverall survival (OS). “Preventing,” “prophylaxis,” or “prevention” of adisease or disorder as used in the context of this invention refers tothe administration of an immunomodulatory polypeptide or engineeredcells of the invention, either alone or in combination with anothercompound, to prevent the occurrence or onset of a disease or disorder orsome or all of the symptoms of a disease or disorder or to lessen thelikelihood of the onset of a disease or disorder.

The term “tumor specific antigen” or “TSA” as used herein refers to acounter-structure that is present primarily on tumor cells of amammalian subject but generally not found on normal cells of themammalian subject. A tumor specific antigen need not be exclusive totumor cells but the percentage of cells of a particular mammal that havethe tumor specific antigen is sufficiently high or the levels of thetumor specific antigen on the surface of the tumor are sufficiently highsuch that it can be targeted by anti-tumor therapeutics, such asimmunomodulatory polypeptides of the invention, and provide preventionor treatment of the mammal from the effects of the tumor. In someembodiments, in a random statistical sample of cells from a mammal witha tumor, at least 50% of the cells displaying a TSA are cancerous. Inother embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% of thecells displaying a TSA are cancerous.

The term “variant” (also “modified” or mutant”) as used in reference toa variant ICOSL means an ICOSL, such as a mammalian (e.g., human ormurine) ICOSL created by human intervention. The variant ICOSL is apolypeptide having an altered amino acid sequence, relative to areference (e.g. unmodified) or wild-type ICOSL. The variant ICOSL is apolypeptide which differs from a reference ICOSL, such as a wild-typeICOSL isoform sequence, by one or more modifications, such as one ormore amino acid substitutions, deletions, additions, or combinationsthereof. For purposes herein, the variant ICOSL contains at least oneaffinity modified domain, whereby one or more of the amino aciddifferences occurs in an IgSF domain (e.g. IgV domain). A variant ICOSLcan contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino aciddifferences, such as amino acid substitutions. A variant ICOSLpolypeptide generally exhibits at least 50%, 60%, 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to a corresponding reference (e.g. unmodified ICOSL)or wild-type, such as to the sequence of SEQ ID NO:5, a mature sequencethereof or a portion thereof containing the extracellular domain or anIgSF domain thereof. In some embodiments, a variant ICOSL polypeptideexhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to acorresponding reference (e.g. unmodified) or wild-type ICOSL, such as areference ICOSL set forth in SEQ ID NO:32 or SEQ ID NOs: 196 or 545.Non-naturally occurring amino acids as well as naturally occurring aminoacids are included within the scope of permissible substitutions oradditions. A variant ICOSL is not limited to any particular method ofmaking and includes, for example, de novo chemical synthesis, de novorecombinant DNA techniques, or combinations thereof. A variant ICOSL ofthe invention specifically binds to CD28, ICOS, and/or CTLA-4 of amammalian species. In some embodiments, the altered amino acid sequenceresults in an altered (i.e., increased or decreased) binding affinity oravidity to ICOS and/or CD28 compared to the reference (e.g. unmodified)or wild-type ICOSL protein. An increase or decrease in binding affinityor avidity can be determined using well known binding assays such asflow cytometry. Larsen et al., American Journal of Transplantation, Vol5: 443-453 (2005). See also, Linsley et al., Immunity, Vol. 1(9):793-801 (1994). An increase in variant ICOSL binding affinity or avidityto ICOS and/or CD28 is to a value at least 5% greater than that of thereference (e.g. unmodified) or wild-type ICOSL and in some embodiments,at least 10%, 15%, 20%, 30%, 40%, 50%, 100% greater than that of thereference (e.g. unmodified) or wild-type ICOSL control value. A decreasein ICOSL binding affinity or avidity to ICOS and/or CD28 is to a valueno greater than 95% of the of the wild-type control values, and in someembodiments no greater than 80%, 70% 60%, 50%, 40%, 30%, 20%, 10%, 5%,or no detectable binding affinity or avidity of the wild-type ICOSand/or CD28 control values. A variant ICOSL is altered in primary aminoacid sequence by substitution, addition, or deletion of amino acidresidues. The term “variant” in the context of variant ICOSL is not beconstrued as imposing any condition for any particular startingcomposition or method by which the variant ICOSL is created. A variantICOSL can, for example, be generated starting from a reference ICOSL orwild type mammalian ICOSL sequence information, then modeled in silicofor binding to ICOS and/or CD28, and finally recombinantly or chemicallysynthesized to yield a variant ICOSL of the present invention. In butone alternative example, a variant ICOSL can be created by site-directedmutagenesis of a reference (e.g. unmodified) or wild-type ICOSL. Thus,variant ICOSL denotes a composition and not necessarily a productproduced by any given process. A variety of techniques includingrecombinant methods, chemical synthesis, or combinations thereof, may beemployed.

The term “wild-type” or “natural” or “native” as used herein is used inconnection with biological materials such as nucleic acid molecules,proteins (e.g., ICOSL), IgSF members, host cells, and the like, refersto those which are found in nature and not modified by humanintervention.

II. VARIANT ICOSL POLYPEPTIDES

Provided herein are variant ICOSL polypeptides that exhibit altered(increased or decreased) binding activity or affinity for one or more ofan ICOSL cognate binding partner. In some embodiments, the ICOSL cognatebinding partner is one or more of CD28, ICOS, or CTLA-4. In someembodiments, the variant ICOSL polypeptide contains one or more aminoacids modifications, such as one or more substitutions (alternatively,“mutations” or “replacements”), deletions or addition, in animmunoglobulin superfamily (IgSF) domain (IgD) relative to a wild-typeor unmodified ICOSL polypeptide or a portion of a wild-type orunmodified ICOSL containing an immunoglobulin superfamily (IgSF) domainor a specific binding fragment thereof. Thus, a provided variant ICOSLpolypeptide is or comprises a variant IgD (hereinafter called “vIgD”) inwhich the one or more amino acid modifications (e.g. substitutions) isin an IgD.

In some embodiments, the IgD comprises an IgV domain or an IgC (e.g.IgC2) domain or specific binding fragment of the IgV domain or the IgC(e.g. IgC2) domain, or combinations thereof. In some embodiments, theIgD can be an IgV only, the combination of the IgV and IgC, includingthe entire extracellular domain (ECD), or any combination of Ig domainsof ICOSL. Table 2 provides exemplary residues that correspond to IgV orIgC regions of ICOSL. In some embodiments, the variant ICOSL polypeptidecontains an IgV domain or an IgC domain or specific binding fragmentsthereof in which the at least one of the amino acid modifications (e.g.substitutions) is in the IgV domain or IgC domain or a specific bindingfragment thereof. In some embodiments, by virtue of the altered bindingactivity or affinity, the IgV domain or IgC domain is anaffinity-modified IgSF domain.

In some embodiments, the variant is modified in one more IgSF domainsrelative to the sequence of a reference (e.g., unmodified) ICOSLsequence. In some embodiments, the reference (e.g., unmodified) ICOSLsequence is a wild-type ICOSL. In some embodiments, the reference (e.g.,unmodified) or wild-type ICOSL has the sequence of a native ICOSL or anortholog thereof. In some embodiments, the reference (e.g., unmodified)or wild-type ICOSL is or comprises the extracellular domain (ECD) ofICOSL or a portion thereof containing one or more IgSF domain (see Table2). In some embodiments, the extracellular domain of a reference (e.g.,unmodified) or wild-type ICOSL polypeptide comprises an IgV domain andan IgC domain or domains. However, the variant ICOSL polypeptide neednot comprise both the IgV domain and the IgC domain or domains. In someembodiments, the variant ICOSL polypeptide comprises or consistsessentially of the IgV domain or a specific binding fragment thereof. Insome embodiments, the variant ICOSL polypeptide comprises or consistsessentially of the IgC domain or specific binding fragments thereof. Insome embodiments, the variant ICOSL is soluble and lacks a transmembranedomain. In some embodiments, the variant ICOSL further comprises atransmembrane domain and, in some cases, also a cytoplasmic domain.

In some embodiments, the reference (e.g., unmodified) or wild-type ICOSLsequence is a mammalian ICOSL sequence. In some embodiments, thereference (e.g., unmodified) or wild-type ICOSL sequence can be amammalian ICOSL that includes, but is not limited to, human, mouse,cynomolgus monkey, or rat. In some embodiments, the reference (e.g.,unmodified) or wildtype ICOSL sequence is human.

In some embodiments, the reference (e.g., unmodified) or wild-type ICOSLsequence has (i) the sequence of amino acids set forth in SEQ ID NO:5 ora mature form thereof lacking the signal sequence, (ii) a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQID NO:5 or the mature form thereof, or (iii) is a portion of (i) or (ii)containing an IgV domain or IgC domain or specific binding fragmentsthereof.

In some embodiments, the reference ICOSL sequence is or comprises anextracellular domain of the ICOSL or a portion thereof. In someembodiments, the reference or wild-type ICOSL polypeptide comprises theamino acid sequence set forth in SEQ ID NO:32, or an ortholog thereof.

(SEQ ID NO: 32) DTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIENVLLQQNLTVGSQTGNDIGERDKITENPVSTGEKNAAT

In some cases, the reference (e.g., unmodified) or wild-type ICOSLpolypeptide can comprise (i) the sequence of amino acids set forth inSEQ ID NO:32, (ii) a sequence of amino acids that has at least about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% sequence identity to SEQ ID NO: 32, or (iii) is a specific bindingfragment of the sequence of (i) or (ii) comprising an IgV domain or anIgC domain.

In some embodiments, the reference ICOSL polypeptide comprises atruncated extracellular domain comprising a C-terminal truncation withreference to the reference ICOSL extracellular domain sequence set forthin SEQ ID NO:32. In some embodiments, the C-terminal truncation is of atleast 10, at least 20, at least 30, at least 40, at least 50, at least60, at least 70, at least 80, at least 90, at least 100, at least 125amino acid residues. In some embodiments, the C-terminal truncation isof at least 1, at least 5 at least 10, at least 15, at least 20, atleast 25, at least 30, at least 35 amino acid residues. In someembodiments, the ICOSL polypeptide comprising a C-terminal truncationdoes not contain, beyond the C-terminus of the truncation point,contiguous amino acid residues of a wild-type ICOSL. Hence, amongprovided ICOSL reference sequences are those that are shorter than thefull extracellular domain of a wild-type ICOSL, e.g. set forth in SEQ IDNO: 32. In some embodiments, the ICOSL polypeptide comprising aC-terminal truncation does not contain or is not fused to amino acidresidues of an ICOSL domain beyond the extracellular domain.

In some embodiments, the ICOSL reference polypeptide is altered, such asmutated or deleted, in one or more protease cleavage site. As foundherein, wild-type ICOSL polypeptide contains a protease cleavage sitethat, in some cases, results in cleavage of the protein upon expressionin cells, e.g. Chinese Hamster Ovary cells, thereby resulting in aheterogeneous product of multiple species, including species ofdifferent lengths or sizes. For example, cleavage of the ICOSLpolypeptide may occur at the LQQN/LT protease cleavage site betweenresidues 207 and 208 of SEQ ID NO: 32 (“/” indicates potential cleavagesite). In some embodiments, the ICOSL reference polypeptide is alteredin or lacks a protease cleavage site set forth as amino acids 204-209 ofSEQ ID NO:32. In some embodiments, a truncated ICOSL polypeptide is moreresistant to protease cleavage compared to a wild-type or non-truncatedICOSL polypeptide. Exemplary truncated ICOSL polypeptide ECD truncationslacking all or a portion of the LQQN/LT protease cleavage site(designated Truncations #2, #3, #4, #5, #6, #7, or #8) are provided inSEQ ID NOs: 600-606.

Truncation #2: (SEQ ID NO: 600)DTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIE NVLLQQNLTruncation #3: (SEQ ID NO: 601)DTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIE NVLLQQNLTVGSQTruncation #4: (SEQ ID NO: 602)DTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIE NVLLQQNTruncation #5: (SEQ ID NO: 603)DTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIE NVLLQQ Truncation #6:(SEQ ID NO: 604) DTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIE NVLL Truncation #7:(SEQ ID NO: 605) DTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIE N Truncation #8:(SEQ ID NO: 606) DTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIE NVLLQQNLT

In some embodiments, the ICOSL reference polypeptide is altered in oneor more amino acids corresponding to amino acids 204-209 with referenceto SEQ ID NO:32. In some embodiments, the variant ICOSL polypeptide hasone or more amino acid modification, e.g., substitution in a referenceICOSL or specific binding fragment thereof corresponding to position(s)207 and/or 208 with reference to numbering of SEQ ID NO:32. In someembodiments, the variant ICOSL polypeptide has one or more amino acidmodification, e.g., substitution, selected from N207A, N207G, L208G, ora conservative amino acid modification, e.g., substitution thereof. Insome embodiments, the one or more amino acid modification, e.g.,substitution is N207A/L208G or N207G/L208G. In some embodiments, thefull length reference ECDs or truncated reference ECDs of the variantICOSL polypeptide are modified to contain one or more amino acidmodifications, e.g., substitutions, selected from N207A, N207G, L208G,or a conservative amino acid modification. Exemplary full length ortruncated reference ECDs with one or more modifications are set forth inSEQ ID NOs: 607-628. Exemplary reference sequences containing mutationsat cleavage site N207 and/or L208 with reference to positions are setforth in SEQ ID NO: 32 are set forth in SEQ ID NOs: 624-628. In somecases, the provided modifications may reduce protease cleavage of theICOSL polypeptide, such as cleavage that may occur at the LQQN/LTprotease cleavage site.

In some embodiments, combinations of the above truncation andmodification strategies can be employed in a reference ICOSL ECDsequence. In some embodiments, the modifications e.g., substitutions,are made in a truncated reference ICOSL polypeptide such as exemplaryreference ICOSL sequence set forth in SEQ ID NOs: 600-606. Exemplaryvariant ICOSL polypeptide sequences with modifications at the potentialprotease cleavage site(s) N207 and/or L208 are set forth in SEQ ID NOs:607-628. In some embodiments, the variant ICOSL polypeptide exhibitsdecreased protease cleavage compared to wild-type ICOSL polypeptide,such as containing the ECD sequence set forth in SEQ ID NO:32.

In some embodiments, the reference (e.g., unmodified) or wildtype ICOSLpolypeptide comprises an IgV domain or an IgC domain, or a specificbinding fragment thereof. In some embodiments, an ICOSL referencepolypeptide containing an IgV domain comprises the amino acid sequenceset forth in SEQ ID NO: 196 (corresponding to amino acid residues 19-129of SEQ ID NO:5), or an ortholog thereof.

(SEQ ID NO: 196) DTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQ SLGFQEVLSVE

In some embodiments, the reference ICOSL polypeptide containing the IgVdomain contains at least amino acids 1-112, 1-113, 1-114, 1-115, 1-116,1-117, 1-118, 1-119, 1-120, 1-121, 1-122, with reference to numberingset forth in SEQ ID NO:32. In some embodiments, an ICOSL referencepolypeptide containing an IgV domain comprises the amino acid sequenceset forth in SEQ ID NO: 545 (corresponding to amino acid residues 19-140of SEQ ID NO:5), or an ortholog thereof. In some embodiments, the IgVdomain is the only IgSF domain of the ICOSL reference polypeptide.

(SEQ ID NO: 545) DTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSV

In some embodiments, the IgV domain of the reference (e.g., unmodified)or wild-type ICOSL polypeptide can contain (i) the sequence of aminoacids set forth in SEQ ID NO: 196 or 545, (ii) a sequence of amino acidsthat has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 196 or 545,or (iii) a specific binding fragment of the sequence of amino acids setforth in SEQ ID NO: 196 or 545 or a specific binding fragment of asequence of (i) or (ii). In some embodiments, the reference (e.g.,unmodified) IgV domain is capable of binding one or more ICOSL cognatebinding proteins, such as one or more of CD28, ICOS, or CTLA-4.

In some embodiments, the IgC domain of the reference (e.g., unmodified)or wild-type ICOSL polypeptide comprises the amino acid sequence setforth as residues 141-227 of SEQ ID NO: 5, or an ortholog thereof. Forexample, the IgC domain of the reference (e.g., unmodified) or wild-typeICOSL polypeptide can contain (i) the sequence of amino acids set forthresidues 141-227 of SEQ ID NO: 5, (ii) a sequence of amino acids thathas at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% sequence identity to residues 141-227 of SEQ IDNO: 5, or (iii) (i) or (ii). In some embodiments, the reference IgVdomain is capable of binding one or more ICOSL cognate binding proteins.

In some embodiments, the reference (e.g., unmodified) or wild-type ICOSLpolypeptide contains a specific binding fragment of ICOSL, such as aspecific binding fragment of the IgV domain or the IgC domain. In someembodiments the specific binding fragment can bind CD28, ICOS, and/orCTLA-4. The specific binding fragment can have an amino acid length ofat least 50 amino acids, such as at least 60, 70, 80, 90, 100, or 110amino acids. In some embodiments, the specific binding fragment of theIgV domain contains an amino acid sequence that is at least about 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ofthe length of the IgV domain set forth as amino acids 19-129 of SEQ IDNO: 5. In some embodiments, the specific binding fragment of the IgCdomain comprises an amino acid sequence that is at least about 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of thelength of the IgC domain set forth as amino acids 141-227 of SEQ ID NO:5.

In some embodiments, the variant ICOSL polypeptide comprises the ECDdomain, a truncated ECD domain, or a portion thereof comprising one ormore affinity modified IgSF domains. In some embodiments, the variantICOSL polypeptides can comprise an IgV domain or an IgC domain, in whichone or more of the IgSF domains (IgV or IgC) or a specific bindingfragment of the IgV domain or a specific binding fragment of the IgCdomain contains the one or more amino acid modifications (e.g.substitutions). In some embodiments, the variant ICOSL polypeptides cancomprise an IgV domain and an IgC domain, or a specific binding fragmentof the IgV domain and a specific binding fragment of the IgC domain. Insome embodiments, the variant ICOSL polypeptide comprises a full-lengthIgV domain. In some embodiments, the variant ICOSL polypeptide comprisesa full-length IgC domain. In some embodiments, the variant ICOSLpolypeptide comprises a specific binding fragment of the IgV domain. Insome embodiments, the variant ICOSL polypeptide comprises a specificbinding fragment of the IgC domain. In some embodiments, the variantICOSL polypeptide comprises a full-length IgV domain and a full-lengthIgC domain. In some embodiments, the variant ICOSL polypeptide comprisesa full-length IgV domain and a specific binding fragment of an IgCdomain. In some embodiments, the variant ICOSL polypeptide comprises aspecific binding fragment of an IgV domain and a full-length IgC domain.In some embodiments, the variant ICOSL polypeptide comprises a specificbinding fragment of an IgV domain and a specific binding fragment of anIgC domain.

In any of such embodiments, the one or more amino acid modifications(e.g., substitutions) of the variant ICOSL polypeptides can be locatedin any one or more of the ICOSL polypeptide domains. For example, insome embodiments, one or more amino acid substitutions are located inthe extracellular domain (ECD) of the variant ICOSL polypeptide, such asset forth in SEQ ID NO: 32. In some embodiments, one or more amino acidsubstitutions are located in the IgV domain or specific binding fragmentof the IgV domain. In some embodiments, one or more amino acidmodifications (e.g. substitutions) are located in the IgC domain orspecific binding fragment of the IgC domain.

Generally, each of the various attributes of polypeptides are separatelydisclosed below (e.g., soluble, secretable and membrane boundpolypeptides, affinity of ICOSL for CD28, ICOS, and CTLA-4, number ofvariations per polypeptide chain, number of linked polypeptide chains,the number and nature of amino acid alterations per variant ICOSL,etc.). However, as will be clear to the skilled artisan, any particularpolypeptide can comprise a combination of these independent attributes.It is understood that reference to amino acids, including to a specificsequence set forth as a SEQ ID NO used to describe domain organizationof an IgSF domain are for illustrative purposes and are not meant tolimit the scope of the embodiments provided. It is understood thatpolypeptides and the description of domains thereof are theoreticallyderived based on homology analysis and alignments with similarmolecules. Thus, the exact locus can vary, and is not necessarily thesame for each protein. Hence, the specific IgSF domain, such as specificIgV domain or IgC domain, can be several amino acids (such as one, two,three or four) longer or shorter.

Further, various embodiments of the invention as discussed below arefrequently provided within the meaning of a defined term as disclosedabove. The embodiments described in a particular definition aretherefore to be interpreted as being incorporated by reference when thedefined term is utilized in discussing the various aspects andattributes described herein. Thus, the headings, the order ofpresentation of the various aspects and embodiments, and the separatedisclosure of each independent attribute is not meant to be a limitationto the scope of the present disclosure.

A. Exemplary Modifications

Provided herein are variant ICOSL polypeptides containing at least oneaffinity-modified IgSF domain (e.g., IgV or IgC) or a specific bindingfragment thereof in an IgSF domain contained in a reference (e.g.,unmodified) or wild-type ICOSL polypeptide such that the variant ICOSLpolypeptide exhibits altered (increased or decreased) binding activityor affinity for one or more ligands ICOS, CD28, or CTLA-4 compared to areference (e.g., unmodified) or wild-type ICOSL polypeptide. In someembodiments, a variant ICOSL polypeptide has a binding affinity forCD28, ICOS, and/or CTLA-4 that differs from that of a reference (e.g.,unmodified) or wild-type ICOSL polypeptide control sequence asdetermined by, for example, solid-phase ELISA immunoassays, flowcytometry or Biacore assays. In some embodiments, the variant ICOSLpolypeptide has an increased binding affinity for CD28, ICOS, and/orCTLA-4. In some embodiments, the variant ICOSL polypeptide has adecreased binding affinity for CD28, ICOS, and/or CTLA-4, relative to areference (unmodified) or wild-type ICOSL polypeptide. The CD28, ICOSand/or the CTLA-4 can be a mammalian protein, such as a human protein ora murine protein.

Binding affinities for each of the cognate binding partners areindependent; that is, in some embodiments, a variant ICOSL polypeptidehas an increased binding affinity for one, two or three of CD28, ICOS,and/or CTLA-4, and a decreased binding affinity for one, two or three ofCD28, ICOS, and CTLA-4, relative to a reference (e.g., unmodified) orwild-type ICOSL polypeptide.

In some embodiments, the variant ICOSL polypeptide has an increasedbinding affinity for CD28, relative to a reference (e.g., unmodified) orwildtype ICOSL polypeptide. In some embodiments, the variant ICOSLpolypeptide has an increased binding affinity for ICOS, relative to areference (e.g., unmodified) or wild-type ICOSL polypeptide. In someembodiments, the variant ICOSL polypeptide has an increased bindingaffinity for CTLA-4, relative to a reference (e.g., unmodified) orwild-type ICOSL polypeptide. In some embodiments, the variant ICOSLpolypeptide has a decreased binding affinity for CD28, relative to areference (e.g., unmodified) or wild-type ICOSL polypeptide. In someembodiments, the variant ICOSL polypeptide has a decreased bindingaffinity for ICOS, relative to a reference (e.g., unmodified) orwild-type ICOSL polypeptide. In some embodiments, the variant ICOSLpolypeptide has a decreased binding affinity for CTLA-4, relative to areference (e.g., unmodified) or wild-type ICOSL polypeptide.

In some embodiments, the variant ICOSL polypeptide has an increasedbinding affinity for CD28 and ICOS, relative to a reference (e.g.,unmodified) or wild-type ICOSL polypeptide. In some embodiments, thevariant ICOSL polypeptide has an increased binding affinity for CD28 anda decreased binding affinity for ICOS, relative to a reference (e.g.,unmodified) or wild-type ICOSL polypeptide. In some embodiments, thevariant ICOSL polypeptide has a decreased binding affinity for CD28 andICOS, relative to a reference (e.g., unmodified) or wild-type ICOSLpolypeptide. In some embodiments, the variant ICOSL polypeptide has adecreased binding affinity for CD28 and an increased binding affinityfor ICOS, relative to a reference (e.g., unmodified) or wild-type ICOSLpolypeptide.

In some embodiments, the variant ICOSL polypeptide has an increasedbinding affinity for CD28 and CTLA-4, relative to a reference (e.g.,unmodified) or wild-type ICOSL polypeptide. In some embodiments, thevariant ICOSL polypeptide has an increased binding affinity for CD28 anda decreased binding affinity for CTLA-4, relative to a reference (e.g.,unmodified) or wild-type ICOSL polypeptide. In some embodiments, thevariant ICOSL polypeptide has a decreased binding affinity for CD28 andCTLA-4, relative to a reference (e.g., unmodified) or wild-type ICOSLpolypeptide. In some embodiments, the variant ICOSL polypeptide has adecreased binding affinity for CD28 and an increased binding affinityfor CTLA-4, relative to a reference (e.g., unmodified) or wild-typeICOSL polypeptide.

In some embodiments, the variant ICOSL polypeptide has an increasedbinding affinity for ICOS and CTLA-4, relative to a reference (e.g.,unmodified) or wild-type ICOS polypeptide. In some embodiments, thevariant ICOSL polypeptide has an increased binding affinity for ICOS anda decreased binding affinity for CTLA-4, relative to a reference (e.g.,unmodified) or wild-type ICOSL polypeptide. In some embodiments, thevariant ICOSL polypeptide has a decreased binding affinity for ICOS andCTLA-4, relative to a reference (e.g., unmodified) or wild-type ICOSLpolypeptide. In some embodiments, the variant ICOSL polypeptide has adecreased binding affinity for ICOS and an increased binding affinityfor CTLA-4, relative to a reference (e.g., unmodified) or wild-typeICOSL polypeptide.

In some embodiments, the variant ICOSL polypeptide has an increasedbinding affinity for CD28, ICOS, and CTLA-4, relative to a reference(e.g., unmodified) or wild-type ICOSL polypeptide. In some embodiments,the variant ICOSL polypeptide has an increased binding affinity for CD28and ICOS, and a decreased binding affinity for CTLA-4, relative to areference (e.g., unmodified) or wild-type ICOSL polypeptide. In someembodiments, the variant ICOSL polypeptide has an increased bindingaffinity for CD28 and CTLA-4, and a decreased binding affinity for ICOS,relative to a reference (e.g., unmodified) or wild-type ICOSLpolypeptide. In some embodiments, the variant ICOSL polypeptide has adecreased binding affinity for CD28 and ICOS, and an increased bindingaffinity for CTLA-4, relative to a reference (e.g., unmodified) orwild-type ICOSL polypeptide. In some embodiments, the variant ICOSLpolypeptide has a decreased binding affinity for CD28 and an increasedbinding affinity for ICOS and CTLA-4, relative to a reference (e.g.,unmodified) or wild-type ICOSL polypeptide. In some embodiments, thevariant ICOSL polypeptide has an increased binding affinity for CD28,and a decreased binding affinity for ICOS and CTLA-4, relative to areference (e.g., unmodified) or wild-type ICOSL polypeptide. In someembodiments, the variant ICOSL polypeptide has a decreased bindingaffinity for CD28, CTLA-4, and ICOS, relative to a reference (e.g.,unmodified) or wild-type ICOSL polypeptide. In some embodiments, thevariant ICOSL polypeptide has a decreased binding affinity for CD28, andan increased binding affinity for ICOS and CTLA-4, relative to areference (e.g., unmodified) or wild-type ICOSL polypeptide.

In some embodiments, a variant ICOSL polypeptide with increased orgreater binding affinity to CD28, ICOS, and/or CTLA-4 will have anincrease in binding affinity relative to the reference (e.g.,unmodified) or wild-type ICOSL polypeptide control of at least about 5%,such as at least about 10%, 15%, 20%, 25%, 35%, or 50% for the CD28,ICOS, and/or CTLA-4. In some embodiments, the increase in bindingaffinity relative to the reference (e.g., unmodified) or wild-type ICOSLpolypeptide is more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold40-fold or 50-fold. In such examples, the reference (e.g., unmodified)or wild-type ICOSL polypeptide has the same sequence as the variantICOSL polypeptide except that it does not contain the one or more aminoacid modifications (e.g., substitutions).

In some embodiments, a variant ICOSL polypeptide with reduced ordecreased binding affinity to CD28, ICOS, and/or CTLA-4 will havedecrease in binding affinity relative to the reference (e.g.,unmodified) or wild-type ICOSL polypeptide control of at least 5%, suchas at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% ormore for the CD28, ICOSL, and/or CTLA-4. In some embodiments, thedecrease in binding affinity relative to the reference (e.g.,unmodified) or wild-type ICOSL polypeptide is more than 1.2-fold,1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 30-fold 40-fold or 50-fold. In such examples,the reference (e.g., unmodified) or wild-type ICOSL polypeptide has thesame sequence as the variant ICOSL polypeptide except that it does notcontain the one or more amino acid modifications, e.g. substitutions.

In some embodiments, the equilibrium dissociation constant (K_(d)) ofany of the foregoing embodiments to CD28, ICOS, and/or CTLA-4 can beless than 1×10⁻⁵ M, 1×10⁻⁶ M, 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M or1×10⁻¹¹ M, or 1×10⁻¹² M.

In some embodiments, a variant ICOSL polypeptide has an increased orgreater binding affinity to CD28. In some embodiments, a variant ICOSLpolypeptide with increased or greater binding affinity to CD28 will havean increase in binding affinity relative to the reference (e.g.,unmodified) or wild-type ICOSL polypeptide control of at least about25%, such as at least about 30%, 40%, 50%, or 60% for CD28. In someembodiments, a variant ICOSL polypeptide with increased or greaterbinding affinity to CD28 has an equilibrium dissociation constant(K_(d)) of less than 200 pM, 300 pM, 400 pM, 500 pM, or 600 pM for CD28.In some embodiments, the variant polypeptide specifically binds to theectodomain of one of ICOS, CD28 or CTLA4 with increased selectivitycompared to the reference (e.g., unmodified) or wild-type ICOSL. In someembodiments, the increased selectivity is for CD28. In some embodiments,the increased selectivity comprises a greater ratio of binding of thevariant ICOSL polypeptide for one cognate binding partner selected fromamong ICOS, CD28 and CTLA4 versus another of the cognate binding partnercompared to the ratio of binding of the reference (e.g., unmodified) orwild-type ICOSL polypeptide for the one cognate binding partner versusthe another of the cognate binding partner. In some embodiments, theratio is greater by at least or at least about 1.5-fold, 2.0-fold,3.0-fold, 4.0-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold,50-fold or more.

The reference (e.g., unmodified) or wild-type ICOSL sequence does notnecessarily have to be used as a starting composition to generatevariant ICOSL polypeptides described herein. Therefore, use of the term“modification”, such as “substitution” does not imply that the presentembodiments are limited to a particular method of making variant ICOSLpolypeptides. Variant ICOSL polypeptides can be made, for example, by denovo peptide synthesis and thus does not necessarily require amodification, such as a “substitution”, in the sense of altering a codonto encode for the modification, e.g. substitution. This principle alsoextends to the terms “addition” and “deletion” of an amino acid residuewhich likewise do not imply a particular method of making. The means bywhich the variant ICOSL polypeptides are designed or created is notlimited to any particular method. In some embodiments, however, areference (e.g., unmodified) or wild-type ICOSL encoding nucleic acid ismutagenized from reference (e.g., unmodified) or wild-type ICOSL geneticmaterial and screened for desired specific binding affinity and/orinduction of IFN-gamma expression or other functional activity. In someembodiments, a variant ICOSL polypeptide is synthesized de novoutilizing protein or nucleic acid sequences available at any number ofpublicly available databases and then subsequently screened. TheNational Center for Biotechnology Information provides such informationand its website is publicly accessible via the internet as is theUniProtKB database as discussed previously.

Unless stated otherwise, as indicated throughout the present disclosure,the amino acid modification (s) are designated by amino acid positionnumber corresponding to the numbering of positions of the reference ECDsequence set forth in SEQ ID NO:32. It is within the level of a skilledartisan to identify the corresponding position of a modification, e.g.amino acid substitution, in an ICOSL polypeptide, including portionthereof containing an IgSF domain (e.g. IgV) thereof, such as byalignment of a reference sequence (e.g. SEQ ID NO: 196, 545, 600-628)with SEQ ID NO:32. In the listing of modifications throughout thisdisclosure, the amino acid position is indicated in the middle, with thecorresponding reference (e.g. unmodified or wild-type) amino acid listedbefore the number and the identified variant amino acid substitutionlisted after the number. If the modification is a deletion of theposition a “del” is indicated and if the modification is an insertion atthe position an “ins” is indicated. In some cases, an insertion islisted with the amino acid position indicated in the middle, with thecorresponding reference amino acid listed before and after the numberand the identified variant amino acid insertion listed after theunmodified (e.g. wild-type) amino acid.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution in a reference (e.g., unmodified)or wild-type ICOSL sequence. The one or more amino acid modification,e.g. substitution, can be in the ectodomain (extracellular domain) ofthe reference (e.g., unmodified) or wild-type ICOSL sequence. In someembodiments, the one or more amino acid modification, e.g. substitutionis in the IgV domain or specific binding fragment thereof. In someembodiments, the one or more amino acid modification, e.g. substitutionis in the IgC domain or specific binding fragment thereof. In someembodiments of the variant ICOSL polypeptide, some of the one or moreamino acid modification, e.g. substitution is in the IgV domain or aspecific binding fragment thereof, and some of the one or more aminoacid modification, e.g. substitution are in the IgC domain or a specificbinding fragment thereof.

In some embodiments, the variant ICOSL polypeptide has up to 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidmodification(s), e.g. substitution. The modification, e.g. substitutioncan be in the IgV domain or the IgC domain. In some embodiments, thevariant ICOSL polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions in theIgV domain or specific binding fragment thereof. In some embodiments,the variant ICOSL polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions inthe IgC domain or specific binding fragment thereof. In someembodiments, the variant ICOSL polypeptide has at least about 85%, 86%,86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity with the reference (e.g., unmodified) or wild-typeICOSL polypeptide or specific binding fragment thereof, such as with theamino acid sequence of SEQ ID NO: 32, 196 or 545.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution in a reference ICOSL or specificbinding fragment there of corresponding to position(s) 10, 11, 13, 16,18, 20, 25, 27, 30, 33, 37, 42, 43, 47, 52, 54, 57, 61, 62, 67, 71, 72,74, 77, 78, 75, 80, 84, 89, 90, 92, 93, 94, 96, 97, 98, 99, 100, 102,103, 107, 109, 110, 111, 113, 115, 116, 117, 119, 120, 121, 122, 126,129, 130, 132, 133, 135, 138, 139, 140, 142, 143, 144, 146, 151, 152,153, 154, 155, 156, 158, 161, 166, 168, 172, 173, 175, 190, 192, 193,194, 198, 201, 203, 207, 208, 210, 212, 217, 218, 220, 221, 224, 225, or227 with reference to numbering of SEQ ID NO:32. In some embodiments,the variant ICOSL polypeptide has one or more amino acid modification,e.g. substitution in a reference ICOSL or specific binding fragmentthere of corresponding to position(s) 10, 11, 13, 16, 18, 20, 25, 26,27, 30, 33, 37, 38, 42, 43, 47, 52, 54, 57, 61, 62, 67, 71, 72, 74, 75,77, 78, 80, 84, 89, 90, 92, 93, 94, 96, 97, 98, 99, 100, 102, 103, 107,109, 110, 111, 113, 115, 116, 117, 119, 120, 121, 122, 126, 129, 130,132, 133, 135, 137, 138, 139, 140, 142, 143, 144, 146, 151, 152, 153,154, 155, 156, 158, 161, 164, 166, 168, 172, 173, 175, 190, 192, 193,194, 198, 201, 203, 207, 208, 210, 212, 217, 218, 220, 221, 224, 225, or227 with reference to numbering of SEQ ID NO:32.

In some embodiments, such variant ICOSL polypeptides exhibit alteredbinding affinity to one or more of CD28, ICOS, and/or CTLA-4 compared tothe reference (e.g., unmodified) or wild-type ICOSL polypeptide. Forexample, in some embodiments, the variant ICOSL polypeptide exhibitsincreased binding affinity to CD28, ICOS, and/or CTLA-4 compared to areference (e.g., unmodified) or wild-type ICOSL polypeptide. In someembodiments, the variant ICOSL polypeptide exhibits decreased bindingaffinity to CD28, ICOS, or CTLA-4 compared to a reference (e.g.,unmodified) or wild-type ICOSL polypeptide.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution selected from M10V, M10I, V11E,S13G, E16V, S18R, A20V, S25G, F27S, F27C, N30D, Y33del, Q37R, K42E,Y47H, T43A, N52A, N52C, N52D, N52G, N52H, N52L, N52K, N52M, N52P, N52Q,N52R, N52S, N52T, N52V, N52Y, S54A, S54P, N57A, N57E, N57F, N57H, N57K,N57L, N57M, N57P, N57Q, N57S, N57T, N57V, N57W, N57Y, R61S, R61C, Y62F,L67P, A71T, G72R, L74Q, R75Q, D77G, F78L, L80P, N84Q, D89G, E90A, K92R,F93L, H94E, H94D, L96F, L96I, V97A, L98F, S99G, Q100A, Q100D, Q100E,Q100G, Q100K, Q100L, Q100M, Q100N, Q100R, Q100P, Q100S, Q100T, Q100V,L102R, G103E, V107A, V107I, S109G, S109N, V110D, V110N, V110A, E111del,T113E, H115R, H115Q, V116A, A117T, N119Q, F120I, F120S, S121G, V122A,V122M, S126T, S126R, H129P, S130G, S132F, Q133H, E135K, F138L, T139S,C140D, C140del, S142F, I143V, 1143T, N144D, Y146C, V151A, Y152C, Y152H,W153R, I154F, N155H, N155Q, K156M, D158G, L161P, L161M, L166Q, N168Q,F172S, L173S, M175T, T190S, T190A, S192G, V193M, N194D, C198R, N201S,L203P, L203F, N207Q, L208P, V210A, S212G, D217V, I218T, I218N, E220G,R221G, R221I, I224V, T225A, N227K or a conservative amino acidmodification, e.g. substitution thereof. In some embodiments, thevariant ICOSL polypeptide has one or more amino acid modification, e.g.substitution selected from M10V, M10I, V11E, S13G, E16V, S18R, A20T,A20V, S25G, R26S, F27C, F27S, N30D, Y33del, Q37R, T38P, K42E, T43A,Y47H, N52A, N52C, N52D, N52G, N52H, N52K, N52L, N52M, N52P, N52Q, N52R,N52S, N52T, N52V, N52Y, S54A, S54F, S54P, N57A, N57D, N57E, N57F, N57H,N57K, N57L, N57M, N57P, N57Q, N57S, N57T, N57V, N57W, N57Y, R61C, R61S,Y62F, L67P, A71T, G72R, L74Q, R75Q, D77G, F78L, L80P, N84Q, D89G, E90A,K92R, F93L, H94D, H94E, L96F, L96I, V97A, L98F, S99G, Q100A, Q100D,Q100E, Q100G, Q100K, Q100L, Q100M, Q100N, Q100P, Q100R, Q100S, Q100T,Q100V, L102R, G103E, V107A, V107I, S109G, S109N, V110A, V110D, V110N,E111del, T113E, H115Q, H115R, V116A, A117T, N119Q, F120I, F120S, S121G,V122A, V122M, S126R, S126T, H129P, S130G, S132F, Q133H, E135K, T137A,F138L, T139S, C140del, C140D, S142F, I143T, I143V, N144D, Y146C, V151A,Y152C, Y152H, W153R, I154F, N155H, N155Q, K156M, D158G, L161M, L161P,Q164L, L166Q, N168Q, F172S, L173S, M175T, T190A, T190S, S192G, V193A,V193M, N194D, C198R, N201S, L203F, L203P, N207Q, L208P, V210A, S212G,D217G, D217V, I218N, I218T, E220G, R221G, R221I, R221K, I224V, T225A,T225S, N227K, or a conservative amino acid substitution thereof.

A conservative amino acid modification, e.g. substitution is any aminoacid that falls in the same class of amino acids as the substitutedamino acids, other than the reference (e.g., unmodified) or wild-typeamino acid. The classes of amino acids are aliphatic (glycine, alanine,valine, leucine, and isoleucine), hydroxyl or sulfur-containing (serine,cysteine, threonine, and methionine), cyclic (proline), aromatic(phenylalanine, tyrosine, tryptophan), basic (histidine, lysine, andarginine), and acidic/amide (aspartate, glutamate, asparagine, andglutamine).

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution selected from M10V, M10I, V11E,S13G, E16V, S18R, A20V, S25G, F27S, F27C, N30D, Y33del, Q37R, K42E,T43A, Y47H, N52A, N52C, N52D, N52G, N52H, N52L, N52K, N52M, N52P, N52Q,N52R, N52S, N52T, N52V, N52Y, S54A, S54P, N57A, N57E, N57F, N57H, N57K,N57L, N57M, N57P, N57Q, N57S, N57T, N57V, N57W, N57Y, R61S, R61C, Y62F,L67P, A71T, G72R, L74Q, R75Q, D77G, F78L, L80P, N84Q, D89G, E90A, K92R,F93L, H94E, H94D, L96F, L96I, V97A, L98F, S99G, Q100A, Q100D, Q100E,Q100G, Q100K, Q100L, Q100M, Q100N, Q100R, Q100P, Q100S, Q100T, Q100V,G103E, L102R, V107A, V107I, S109G, S109N, V110D, V110N, V110A, E111del,T113E, H115R, H115Q, V116A, A117T, N119Q, F120I, F120S, S121G, V122A,V122M, S126T, S126R, H129P, S130G, S132F, Q133H, E135K, F138L, T139S,C140D, C140del, S142F, I143V, I143T, N144D, Y146C, V151A, Y152C, Y152H,W153R, I154F, N155H, N155Q, K156M, D158G, L161P, L161M, L166Q, N168Q,F172S, L173S, M175T, T190A, T190S, S192G, V193M, N194D, C198R, N201S,L203P, L203F, N207Q, L208P, V210A, S212G, D217V, I218T, I218N, E220G,R221G, R221I, I224V, T225A, or N227K. In some embodiments, the variantICOSL polypeptide has one or more amino acid modification, e.g.substitution selected from M10V, M10I, V11E, S13G, E16V, S18R, A20T,A20V, S25G, R26S, F27C, F27S, N30D, Y33del, Q37R, T38P, K42E, T43A,Y47H, N52A, N52C, N52D, N52G, N52H, N52K, N52L, N52M, N52P, N52Q, N52R,N52S, N52T, N52V, N52Y, S54A, S54F, S54P, N57A, N57D, N57E, N57F, N57H,N57K, N57L, N57M, N57P, N57Q, N57S, N57T, N57V, N57W, N57Y, R61C, R61S,Y62F, L67P, A71T, G72R, L74Q, R75Q, D77G, F78L, L80P, N84Q, D89G, E90A,K92R, F93L, H94D, H94E, L96F, L96I, V97A, L98F, S99G, Q100A, Q100D,Q100E, Q100G, Q100K, Q100L, Q100M, Q100N, Q100P, Q100R, Q100S, Q100T,Q100V, L102R, G103E, V107A, V107I, S109G, S109N, V110A, V110D, V110N,E111del, T113E, H115Q, H115R, V116A, A117T, N119Q, F120I, F120S, S121G,V122A, V122M, S126R, S126T, H129P, S130G, S132F, Q133H, E135K, T137A,F138L, T139S, C140del, C140D, S142F, I143T, I143V, N144D, Y146C, V151A,Y152C, Y152H, W153R, I154F, N155H, N155Q, K156M, D158G, L161M, L161P,Q164L, L166Q, N168Q, F172S, L173S, M175T, T190A, T190S, S192G, V193A,V193M, N194D, C198R, N201S, L203F, L203P, N207Q, L208P, V210A, S212G,D217G, D217V, I218N, I218T, E220G, R221G, R221I, R221K, I224V, T225A,T225S, N227K, or a conservative amino acid substitution thereof.

In some embodiments, the one or more amino acid modification, e.g.substitution is N52Y/N57Y/F138L/L203P, N52H/N57Y/Q100P,N52S/Y146C/Y152C, N52H/C198R, N52H/C140D/T225A, N52H/C198R/T225A,N52H/K92R, N52H/S99G, N57Y/Q100P, N52S/G103E, N52S/S130G/Y152C,N52S/Y152C, N52S/C198R, N52Y/N57Y/Y152C, N52Y/N57Y/H129P/C198R,N52H/L161P/C198R, N52S/T113E, N52D/S54P, N52K/L208P, N52S/Y152H,N52D/V151A, N52H/I143T, N52S/L80P, F120S/Y152H/N201S, N52S/R75Q/L203P,N52S/D158G, N52D/Q133H, N52S/N57Y/H94D/L96F/L98F/Q100R,N52S/N57Y/H94D/L96F/L98F/Q100R/G103E/F120S, N52H/F78L/Q100R,N52H/N57Y/Q100R/V110D, N52H/N57Y/R75Q/Q100R/V110D, N52H/N57Y/Q100R,N52H/N57Y/L74Q/Q100R/V110D, N52H/Q100R, N52H/S121G,A20V/N52H/N57Y/Q100R/S109G, N52H/N57Y/Q100P,N52H/N57Y/R61S/Q100R/V110D/L173S, N52H/N57Y/Q100R/V122A,N52H/N57Y/Q100R/F172S, N52H/N57Y, N52S/F120S, N52S/V97A, N52S/G72R,N52S/A71T/A117T, N52S/E220G, Y47H/N52S/V107A/F120S,N52H/N57Y/Q100R/V110D/S132F/M175T,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R,Q37R/N52H/N57Y/Q100R/V110N/S142F/C198R/D217V/R221G,N52H/N57Y/Q100R/V110D/C198R,N52H/N57Y/Q100R/V110D/VD/V116A/L161M/F172S/S192G/C198R,F27S/N52H/N57Y/V110N, N52S/H94E/L96I/S109N/L166Q,S18R/N52S/F93L/I143V/R221G, A20T/N52D/Y146C/Q164L,V11E/N30D/N52H/N57Y/H94E/L96I/L98F/N194D/V210A/I218T,N52S/H94E/L96I/V122M, N52H/N57Y/H94E/L96I/F120I/S126T/W153R/I218N,M10V/S18R/N30D/N52S/S126R/T139S/L203F, S25G/N30D/N52S/F120S/N227K,N30D/N52S/L67P/Q100K/D217G/R221K/T225S,N52H/N57Y/Q100R/V110D/A117T/T190S/C198R,N52H/N57Y/Q100R/V110D/F172S/C198R,S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173S/C198R,N52H/N57Y/V110A/C198R/R221I,M10I/S13G/N52H/N57Y/D77G/V110A/H129P/I143V/F172S/V193M, C198R,N52H/N57Y/R61C/Y62F/Q100R/V110N/F120S/C198R,N52H/N57Y/Q100R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/N144D/F172S/C198R, N52S/H94E/L98F/Q100R,N52S/E90A, N30D/K42E/N52S, N52S/F120S/I143V/I224V,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/S54P, T38P/N52S/N57D,N52H/C140del/T225A, N52H/F78L/Q100R/C198R, N52H/N57Y/R75Q/Q100P/V110D,N52H/N57Y/L74Q/V110D/S192G, N52H/S121G/C198R, N52S/F120S/N227K,N52S/A71T/A117T/T190A/C198R, T43A/N52H/N57Y/L74Q/D89G/V110D/F172S,N52H/N57Y/Q100R/V110D/S132F/M175T,N52H/N57Y/Q100R/V107I/V110D/I154F/C198R/R221G, Q100R, F138L/L203P,N57Y/F138L/L203P, N57Y/Q100R/C198R, N57Y/F138L/L203P, Q100R/F138L,L203P, N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R,N52H/N57Y/Q100R/L102R/H15R/F72S/C198R, N52H/V122A/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/N194D, N52H/N57Y/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/H115R, N52H/N57Y/Q100R/H115R,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/F172S, N52H/Q100R/H115R/I143T/F172S,N52H/N57Y/Q100P/H115R/F172S, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/E90A/H115R, N30D/K42E N52S/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N52S/H115R/F120S/I143V/C198R, N52S/H115R/F172S/C198R,N52H/N57Y/Q100P/C198R, N52H/N57Y/Q100P H115R/F172S/C198R,N52H/N57Y/Q100P/F172S/C198R, N52H/N57Y/Q100P/H115R,N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R, N52H/Q100R/H115R/F172S,N52H/Q100R/F172S/C198R, N52H/Q100R/H115Q/F172S/C198R,N52H/N57Y/Q100R/F172S/C198R, N52Q/N207Q, N168Q/N207Q, N52Q/N168Q,N84Q/N207Q, N155Q/N207Q, N119Q/N168Q, N119Q/N207Q, N119Q/N155Q,N52Q/N84Q, N52Q/N119Q, N84Q/N119Q, N52Q/N84Q/N168Q, N52Q/N84Q/N207Q,N84Q/N155Q/N168Q, N84Q/N168Q/N207Q, N84Q/N155H/N207Q, N155Q/N168Q/N207Q,N119Q/N155Q/N168Q, N119Q/N168Q/N207Q, N84Q/N119Q/N207Q,N119Q/N155H/N207Q, N84Q/N119Q/N155Q, N52Q/N119Q/N155Q, N52H/N84Q,N52H/N84Q/N168Q, N52H/N84Q/N207Q, N52H/N84Q/N168Q/N207Q,N52Q/N84Q/N155Q, N52Q/N84Q/N168Q, N52Q/N84Q/N155Q/N168Q,N52Q/N84Q/N119Q/N168Q, N84Q/N119Q/N155Q/N168Q, N84Q/N155Q/N168Q/N207Q,N84Q/N119Q/N155Q/N207Q, N52Q/N84Q/N119Q/N207Q, N52Q/N84Q/N119Q/N155Q,N52Q/N84Q/N119Q/N155Q/N207Q, N84Q/N119Q/N155Q/N168Q/N207Q,N52A/N57F/Q100S, N52A/N57H/Q100S, N52A/N57Y/Q100A, N52D/N57A/Q100A,N52D/Q100S, N52G/Q100A, N52H/Q100A, N52M/N57H/Q100S, N52M/N57W/Q100P,N52Q/N57F, N52Q/N57S/Q100A, N52R/N57L/Q100A, N52R/N57Y/Q100P,N52R/N57Y/Q100S, N52S/N57A/Q100A, N52S/N57H/Q100E, N52S/N57L/Q100S,N52S/N57M/Q100S, N52S/N57Y/Q100S, N52S/N57Y/Q100M, N52S/N57Y/Q100V,N52T/N57H/Q100S, N52T/N57H/Q100A, N52T/N57Y/Q100A, N52V/N57L/Q100A,N52H/N57Y/Q100K, N52K/N57Y/Q100R, N52L/N57H/Q100R, N52R/N57F/Q100N,N52R/N57F/Q100P, N52R/N57F/Q100R, N52R/N57F/Q100T, N52R/N57H/Q100K,N52R/N57L/Q100S, N52R/N57W/Q100K, N52R/N57W, N52R/N57Y/Q100R,N52C/N57E/Q100S, N52G/N57P/Q100D, N52G/N57V/Q100G, N52G/N57V, N52L/N57V,N52P/N57P, N52P/N57S/Q100G, N52S/N57L/Q100G, N52T/N57K/Q100P,N52V/N57T/Q100L, or N57Q/Q100P.

In some embodiments, the one or more amino acid modifications areselected from among N52Y/N57Y/F138L/L203P, N52H/N57Y/Q100P,N52S/Y146C/Y152C, N52H/C198R, N52H/C140D/T225A, N52H/C198R/T225A,N52H/K92R, N52H/S99G, N57Y/Q100P, N52S/S130G/Y152C, N52S/Y152C,N52S/C198R, N52Y/N57Y/Y152C, N52Y/N57Y/H129P/C198R, N52H/L161P/C198R,N52S/T113E, N52D/S54P, N52K/L208P, N52S/Y152H, N52D/V151A, N52H/I143T,N52S/L80P, F120S/Y152H/N201S, N52S/R75Q/L203P, N52S/D158G, N52D/Q133H,N52S/N57Y/H94D/L96F/L98F/Q100R,N52S/N57Y/H94D/L96F/L98F/Q100R/G103E/F120S, N52S/G103E, N52H/F78L/Q100R,N52H/N57Y/Q100R/V110D, N52H/N57Y/R75Q/Q100R/V110D, N52H/N57Y/Q100R,N52H/N57Y/L74Q/Q100R/V10D, N52H/Q100R, N52H/S121G,A20V/N52H/N57Y/Q100R/S109G, N52H/N57Y/R61S/Q100R/V110D/L173S,N52H/N57Y/Q100R/V122A, N52H/N57Y/Q100R/F172S, N52H/N57Y, N52S/F120S,N52S/V97A, N52S/G72R, N52S/A71T/A117T, N52S/E220G,Y47H/N52S/V107A/F120S, N52H/N57Y/Q100R/V110D/S132F/M175T,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R,Q37R/N52H/N57Y/Q100R/V110N/S142F/C198R/D217V/R221G,N52H/N57Y/Q100R/V110D/C198R,N52H/N57Y/Q100R/V110D/V116A/L161M/F172S/S192G/C198R,F27S/N52H/N57Y/V110N, N52S/H94E/L96I/S109N/L166Q,S18R/N52S/F93L/I143V/R221G, A20T/N52D/Y146C/Q164L,V11E/N30D/N52H/N57Y/H94E/L96I/L98F/N194D/V210A/I218T,N52S/H94E/L96I/V122M, N52H/N57Y/H94E/L96I/F120I/S126T/W153R/I218N,M10V/S18R/N30D/N52S/S126R/T139S/L203F, S25G/N30D/N52S/F120S/N227K,N30D/N52S/L67P/Q100K/D217G/R221K/T225S,N52H/N57Y/Q100R/V110D/A117T/T190S/C198R,N52H/N57Y/Q100R/V110D/F172S/C198R,S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173S/C198R,N52H/N57Y/V110A/C198R/R221I,M10I/S13G/N52H/N57Y/D77G/V110A/H129P/I143V/F172S/V193M, C198R,N52H/N57Y/R61C/Y62F/Q100R/V110N/F120S/C198R,N52H/N57Y/Q100R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/N144D/F172S/C198R, N52S/H94E/L98F/Q100R,N52S/E90A, N30D/K42E/N52S, N52S/F120S/I143V/I224V,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/S54P, T38P/N52S/N57D,N52H/C140del/T225A, N52H/F78L/Q100R/C198R, N52H/N57Y/R75Q/Q100P/V110D,N52H/N57Y/L74Q/V110D/S192G, N52H/S121G/C198R, N52S/F120S/N227K,N52S/A71T/A117T/T190A/C198R, T43A/N52H/N57Y/L74Q/D89G/V110D/F172S,N52H/N57Y/Q100R/V110D/S132F/M175T,N52H/N57Y/Q100R/V107I/V110D/I154F/C198R/R221G, N52Q/N207Q, N168Q/N207Q,N52Q/N168Q, N84Q/N207Q, N155Q/N207Q, N119Q/N168Q, N119Q/N207Q,N119Q/N155Q, N52Q/N84Q, N52Q/N119Q, N84Q/N119Q, N52Q/N84Q/N168Q,N52Q/N84Q/N207Q, N84Q/N155Q/N168Q, N84Q/N7Q, N4Q/N155H/N207Q,N155Q/N168Q/N207Q, N119Q N155Q/N168Q, N119Q/N168Q/N207Q,N84Q/N119Q/N207Q, N119Q/N155H/N207Q, N84Q/N119Q/N155Q, N52Q/N119Q/N155Q,N52H/N84Q/N119Q, N52H/N84Q, N52H/N84Q/N168Q/N207Q,N52Q/N84Q/N155Q/N168Q, N52Q/N84Q/N119Q/N168Q, N84Q/N119Q/N155Q/N168Q,N84Q/N155Q/N168Q/N207Q, N84Q/N119Q/N155Q/N207Q, N52Q/N84Q/N119Q/N207Q,N52Q/N84Q/N19Q/N155Q, N52Q/N4Q/N19Q/N155Q/N207Q,N84Q/N119Q/N155Q/N168Q/N207Q, F138L/L203P, N52Y/F138L/L203P,N57Y/Q100R/C198R, N57Y/F138L/L203P, Q100R/F138L,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R,N52H/N57Y/Q100R/L102R/H115R/F172S/C198R, N52H/V122A/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/N194D, N52H/N57Y/H115R/F172S/C198R,N52H/N57Y/H115R, N52H/N57Y/Q100R/H115R,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/F172S, N52H/Q100R/H115R/I143T/F172S,N52H/N57Y/Q100P/H115R/F172S, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/E90A/H115R, N30D/K42E N52S/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N52S/H115R/F120S/I143V/C198R, N52S/H115R/F172S/C198R,N52H/N57Y/Q100P/C198R, N52H/N57Y/Q100P/H115R/F172S/C198R,N52H/N57Y/Q100P/F172S/C198R, N52H/N57Y/Q100P/H115R,N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R, N52H/Q100R/H115R/F172S,N52H/Q100R/F172S/C198R, N52H/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/F172S/C198R, N52A/N57F/Q100S, N52A/N57H/Q100S,N52A/N57Y/Q100A, N52D/N57A/Q100A, N52D/Q100S, N52G/Q100A, N52H/Q100A,N52M/N57H/Q100S, N52M/N57W/Q100P, N52Q/N57F, N52Q/N57S/Q100A,N52R/N57L/Q100A, N52R/N57Y/Q100P, N52R/N57Y/Q100S, N52S/N57A/Q100A,N52S/N57H/Q100E, N52S/N57L/Q100S, N52S/N57M/Q100S, N52S/N57Y/Q100S,N52S/N57Y/Q100M, N52S/N57Y/Q100V, N52T/N57H/Q100S, N52T/N57H/Q100A,N52T/N57Y/Q100A, N52V/N57L/Q100A, N52H/N57Y/Q100K, N52K/N57Y/Q100R,N52L/N57H/Q100R, N52R/N57F/Q100N, N52R/N57F/Q100P, N52R/N57F/Q100R,N52R/N57F/Q100T, N52R/N57H/Q100K, N52R/N57L/Q100S, N52R/N57W/Q100K,N52R/N57W, N52R/N57Y/Q100R, N52C/N57E/Q100S, N52G/N57P/Q100D,N52G/N57V/Q100G, N52G/N57V, N52L/N57V, N52P/N57P, N52P/N57S/Q100G,N52S/N57L/Q100G, N52T/N57K/Q100P, N52V/N57T/Q100L, N57Q/Q100P,S54F/V193A or R26S/N52H/N57Y/V110D/T137A/C198R.

In some embodiments, the variant ICOSL polypeptide comprises any of themutations listed in Table 1. Table 1 also provides exemplary sequencesby reference to SEQ ID NO for the extracellular domain (ECD) or IgVdomain of the reference (e.g., unmodified) or wild-type ICOSL orexemplary variant ICOSL polypeptides. As indicated, the exact locus orresidues corresponding to a given domain can vary, such as depending onthe methods used to identify or classify the domain. Also, in somecases, adjacent N- and/or C-terminal amino acids of a given domain (e.g.IgV) also can be included in a sequence of a variant IgSF polypeptide,such as to ensure proper folding of the domain when expressed. Thus, itis understood that the exemplification of the SEQ ID NOSs in Table 1 isnot to be construed as limiting. For example, the particular domain,such as the ECD domain, of a variant ICOSL polypeptide can be severalamino acids longer or shorter, such as 1-10, e.g., 1, 2, 3, 4, 5, 6 or 7amino acids longer or shorter, than the sequence of amino acids setforth in the respective SEQ ID NO.

In some embodiments, the variant ICOSL polypeptide comprises any of themutations listed in Table 1. In some examples, the mutations are made ina reference ICOSL containing the sequence of amino acids set forth inSEQ ID NO: 32, a reference ICOSL that contains the IgV domain of ICOSLset forth in SEQ ID NOs: 196 or 545, or a reference ICOSL that istruncated and/or modified containing the sequence of amino acids setforth in any of SEQ ID NOs: 600-628. In some embodiments, the variantICOSL polypeptide comprises any of the extracellular domain (ECD)sequences listed in Table 1 (i.e., any one of SEQ ID NOS: 109-142, 239,280-325, 364-381, 387-424, 427-433, 435-470, 638-685). In someembodiments, the variant ICOSL polypeptide comprises a polypeptidesequence that exhibits at least 90% identity, at least 91% identity, atleast 92% identity, at least 93% identity, at least 94% identity, atleast 95% identity, such as at least 96% identity, 97% identity, 98%identity, or 99% identity to any of the extracellular domain (ECD)sequences listed in Table 1 (i.e., any one of SEQ ID NOS: 109-142, 239,280-325, 364-381, 387-424, 427-433, 435-470, 638-685) and contains theamino acid modification(s), e.g. substitution(s) not present in thereference (e.g., unmodified) or wild-type ICOSL. In some embodiments,the variant ICOSL polypeptide comprises a specific binding fragment ofany of the extracellular domain (ECD) sequences listed in Table 1 (i.e.,any one of SEQ ID NOS: 109-142, 239, 280-325, 364-381, 387-424, 427-433,435-470, 638-685) and contains the amino acid modification(s), e.g.substitution (s) not present in the reference (e.g., unmodified) orwild-type ICOSL.

In some embodiments, the variant ICOSL polypeptide comprises any of theextracellular domain (ECD) sequences listed in Table 1 (i.e., any one ofSEQ ID NOS: 109-142, 239, 280-325, 364-381, 387-424, 427-433, 435-470,638-685, 905, 908). In some embodiments, the variant ICOSL polypeptidecomprises a polypeptide sequence that exhibits at least 90% identity, atleast 91% identity, at least 92% identity, at least 93% identity, atleast 94% identity, at least 95% identity, such as at least 96%identity, 97% identity, 98% identity, or 99% identity to any of theextracellular domain (ECD) sequences listed in Table 1 (i.e., any one ofSEQ ID NOS: 109-142, 239, 280-325, 364-381, 387-424, 427-433, 435-470,638-685, 905, 908) and contains the amino acid modification(s), e.g.substitution(s) not present in the reference (e.g., unmodified) orwild-type ICOSL. In some embodiments, the variant ICOSL polypeptidecomprises a specific binding fragment of any of the extracellular domain(ECD) sequences listed in Table 1 (i.e., any one of SEQ ID NOS: 109-142,239, 280-325, 364-381, 387-424, 427-433, 435-470, 638-685, 905, 908) andcontains the amino acid modification(s), e.g. substitution (s) notpresent in the reference (e.g., unmodified) or wild-type ICOSL.

In some embodiments, the variant ICOSL polypeptide comprises any of theIgV sequences listed in Table 1 (i.e., any one of SEQ ID NOS: 197-199,201-208, 210, 212, 240, 326-340, 382-386, 425-426, 434, 546-599,686-857). In some embodiments, the variant ICOSL polypeptide comprises apolypeptide sequence that exhibits at least 90% identity, at least 91%identity, at least 92% identity, at least 93% identity, at least 94%identity, at least 95% identity, such as at least 96% identity, 97%identity, 98% identity, or 99% identity to any of the IgV sequenceslisted in Table 1 (i.e., any one of SEQ ID NOS: 197-199, 201-208, 210,212, 240, 326-340, 382-386, 425-426, 434, 546-599, 686-857) and containsthe amino acid modification(s), e.g. substitution(s) not present in thereference (e.g., unmodified) or wild-type ICOSL. In some embodiments,the variant ICOSL polypeptide comprises a specific binding fragment ofany of the IgV sequences listed in Table 1 (i.e., any one of SEQ ID NOS:197-199, 201-208, 210, 212, 240, 326-340, 382-386, 425-426, 434,546-599, 686-857) and contains the amino acid substitution(s) notpresent in the reference (e.g., unmodified) or wild-type ICOSL.

In some embodiments, the variant ICOSL polypeptide comprises any of theIgV sequences listed in Table 1 (i.e., any one of SEQ ID NOS: 197-199,201-208, 210, 212, 240, 326-340, 382-386, 425-426, 434, 546-599,686-857, 906-907, 909-910). In some embodiments, the variant ICOSLpolypeptide comprises a polypeptide sequence that exhibits at least 90%identity, at least 91% identity, at least 92% identity, at least 93%identity, at least 94% identity, at least 95% identity, such as at least96% identity, 97% identity, 98% identity, or 99% identity to any of theIgV sequences listed in Table 1 (i.e., any one of SEQ ID NOS: 197-199,201-208, 210, 212, 240, 326-340, 382-386, 425-426, 434, 546-599,686-857, 906-907, 909-910) and contains the amino acid modification(s),e.g. substitution(s) not present in the reference (e.g., unmodified) orwild-type ICOSL. In some embodiments, the variant ICOSL polypeptidecomprises a specific binding fragment of any of the IgV sequences listedin Table 1 (i.e., any one of SEQ ID NOS: 197-199, 201-208, 210, 212,240, 326-340, 382-386, 425-426, 434, 546-599, 686-857, 906-907, 909-910)and contains the amino acid substitution(s) not present in the reference(e.g., unmodified) or wild-type ICOSL.

Mutations designated with an “X” indicate the designated positioncontains a Q or the wild-type residue set forth in the correspondingposition of SEQ ID NO: 32.

TABLE 1 Exemplary variant ICOSL polypeptides ECD IgV SEQ ID SEQ IDMutation(s) NO NO Wild-type 32 196, 545 N52S 109 197, 546 N52H 110 198,547 N52D 111 199, 548 N52Y/N57Y/F138L/L203P 112 782, 783 N52H/N57Y/Q100P113 201, 549 N52S/Y146C/Y152C 114 197, 546 N52H/C198R 115 198, 547N52H/C140D/T225A 116 198, 547 N52H/C198R/T225A 117 198, 547 N52H/K92R118 202, 550 N52H/S99G 119 203, 551 N52Y 120 204, 552 N57Y 121 205, 553N57Y/Q100P 122 206, 554 N52S/S130G/Y152C 123 197, 546 N52S/Y152C 124197, 546 N52S/C198R 125 197, 546 N52Y/N57Y/Y152C 126 782, 783N52Y/N57Y/H129P/C198R 127 782, 783 N52H/L161P/C198R 128 198, 547N52S/T113E 129 197, 555 S54A 130 207, 556 N52D/S54P 131 208, 557N52K/L208P 132 785, 786 N52S/Y152H 133 197, 546 N52D/V151A 134 199, 548N52H/I143T 135 198, 547 N52S/L80P 136 210, 558 F120S/Y152H/N201S 137196, 545 N52S/R75Q/L203P 138 787, 788 N52S/D158G 139 197, 546 N52D/Q133H140 199, 548 N52S/N57Y/H94D/L96F/L98F/ 141 212, 559 Q100RN52S/N57Y/H94D/L96F/L98F/ 142 789, 560 Q100R/G103E/F120S N52S/G103E 239240, 561 N52H/F78L/Q100R 280 326, 562 N52H/N57Y/Q100R/V110D 281 327, 563N52H/N57Y/R75Q/Q100R/V110D 282 328, 564 N52H/N57Y/Q100R 283 329, 565N52H/N57Y/L74Q/Q100R/V110D 284 330, 566 N52H/Q100R 285 331, 567N52H/S121G 286 198, 568 A20V/N52H/N57Y/Q100R/S109G 287 332, 569N52H/N57Y/Q100P 288 333, 570 N52H/N57Y/R61S/Q100R/V110D/ 289 791, 792L173S N52H/N57Y/Q100R/V122A 290 329, 571 N52H/N57Y/Q100R/F172S 291 329,565 N52H/N57Y 292 334, 572 N52S/F120S 293 197, 573 N52S/V97A 294 335,574 N52S/G72R 295 336, 575 N52S/A71T/A117T 296 793, 576 N52S/E220G 297197, 546 Y47H/N52S/V107A/F120S 298 794, 577 N52H/N57Y/Q100R/V110D/S132F/299 327, 563 M175T E16V/N52H/N57Y/Q100R/V110D/ 300 795, 796H115R/Y152C/K156M/C198R Q37R/N52H/N57Y/Q100R/V110N/ 301 797, 798S142F/C198R/D217V/R221G N52H/N57Y/Q100R/V110D/C198R 302 327, 563N52H/N57Y/Q100R/V110D/V116A/ 303 799, 800 L161M/F172S/S192G/C198RF27S/N52H/N57Y/V110N 304 337, 578 N52S/H94E/L96I/S109N/L166Q 305 801,802 S18R/N52S/F93L/I143V/R221G 306 803, 804 A20T/N52D/Y146C/Q164L 307805, 806 V11E/N30D/N52H/N57Y/H94E/ 308 807, 808L96I/L98F/N194D/V210A/I218T N52S/H94E/L96I/V122M 309 809, 579N52H/N57Y/H94E/L96I/F120I/ 310 810, 811 S126T/W153R/I218NM10V/S18R/N30D/N52S/S126R/ 311 812, 813 T139S/L203FS25G/N30D/N52S/F120S/N227K 312 814, 815 N30D/N52S/L67P/Q100K/D217G/ 313816, 817 R22IK/T225S N52H/N57Y/Q100R/V110D/A117T/ 314 327, 818T190S/C198R N52H/N57Y/Q100R/V110D/F172S/ 315 327, 563 C198RS25G/F27C/N52H/N57Y/Q100R/ 316 819, 820 V110D/E135K/L173S/C198RN52H/N57Y/V110A/C198R/R221I 317 821, 822 M10I/S13G/N52H/N57Y/D77G/ 318823, 824 V110A/H129P/I143V/F172S/ V193M/C198R N52H/N57Y/R61C/Y62F/Q100R/319 825, 826 V110N/F120S/C198R N52H/N57Y/Q100R/V110D/H115R/ 320 327, 827C198R N52H/N57Y/Q100R/V110D/N144D/ 321 327, 563 F172S/C198RN52S/H94E/L98F/Q100R 322 338, 580 N52S/E90A 323 339, 581 N30D/K42E/N52S324 340, 582 N52S/F120S/I143V/I224V 325 197, 573N52H/N57Y/Q100R/V110D/C198R/ 364 828, 829 S212G N52H/N57Y/Q100R/C198R365 329, 565 N52S/N194D 366 197, 546 N52H/N57Y/Q100R/L102R/V110D/ 367830, 831 H115R/C198R N52S/S54P 368 382, 583 T38P/N52S/N57D 369 383, 584E111del 370 384, 585 Y33del 371 385, 586 N52H/C140del/T225A 372 198, 547N52H/F78L/Q100R/C198R 373 326, 562 N52H/N57Y/R75Q/Q100P/V110D 374 386,587 N52H/N57Y/L74Q/V110D/S192G 375 832, 833 N52H/S121G/C198R 376 198,568 N52S/F120S/N227K 377 197, 573 N52S/A71T/A117T/T190A/C198R 378 793,576 T43A/N52H/N57Y/L74Q/D89G/ 379 790, 834 V110D/F172SN52H/N57Y/Q100R/V110D/S132F/ 380 327, 563 M175TN52H/N57Y/Q100R/V107I/V110D/ 381 835, 836 I154F/C198R/R221G N84Q 387425, 588 N119Q 388 196, 842 N168Q 389 196, 545 N207Q 390 196, 545N52Q/N207X 391 837, 838 N168X/N207X 392 196, 545 N52Q/N168Q 393 837, 838N84Q/N207Q 394 425, 840 N155Q/N207Q 395 196, 545 N119Q/N168Q 396 196,842 N119Q/N207Q 397 196, 842 N119Q/N155X 398 196, 842 N52Q/N84Q 399 426,590 N52Q/N119Q 400 837, 591 N84Q/N119Q 401 425, 592 N52Q/N84Q/N168Q 402426, 590 N52Q/N84Q/N207Q 403 426, 590 N84Q/N155Q/N168Q 404 425, 588N84Q/N168Q/N207Q 405 425, 588 N84Q/N155H/N207Q 406 425, 588N155Q/N168Q/N207Q 407 196, 545 N119Q/N155Q/N168Q 408 196, 842N119Q/N168Q/N207Q 409 196, 842 N84Q/N119Q/N207Q 410 425, 592N119Q/N155H/N207Q 411 196, 842 N84Q/N119Q/N155Q 412 425, 592N52Q/N119Q/N155Q 413 837, 591 N52H/N84Q/N119Q 414 839, 593N52H/N84Q/N168X/N207X 415 839, 841 N52Q/N84Q/N155X/N168X 416 426, 590N52Q/N84Q/N119Q/N168Q 417 426, 843 N84Q/N119Q/N155Q/N168Q 418 425, 592N84Q/N155Q/N168Q/N207Q 419 425, 588 N84Q/N119Q/N155Q/N207Q 420 425, 592N52Q/N84Q/N119Q/N207Q 421 426, 843 N52Q/N84Q/N119Q/N155Q 422 426, 843N52Q/N84Q/N119Q/N155Q/N207Q 423 426, 843 N84Q/N119Q/N155Q/N168Q/N207Q424 425, 592 Q100R 427 434, 594 F138L/L203P 428 196, 545N52Y/F138L/L203P 429 204, 552 N57Y/Q100R/C198R 430 844, 845N57Y/F138L/L203P 431 205, 553 Q100R/F138L 432 846, 847 L203P 433 196,545 N52H/N57Y/Q100R/H115R/C198R 435 329, 596 N52H/N57Y/Q100R/F172S/C198R436 329, 565 N52H/N57Y/Q100R/H115R/F172S/ 437 329, 596 C198RN52H/N57Y/Q100R/H115R/I143V/ 438 329, 596 F172S/C198RN52H/N57Y/Q100R/L102R/H115R/ 439 849, 850 F172S/C198RN52H/V122A/F172S/C198R 440 198, 851 N52H/N57Y/Q100R/H115R/F172S/ 441329, 596 N194D N52H/N57Y/H115R/F172S/C198R 442 334, 595N52H/N57Y/Q100R/H115R/C198R 443 329, 596 N52H/N57Y/H115R 444 334, 595N52H/N57Y/Q100R/H115R 445 329, 596 N52H/N57Y/Q100R/H115R/F172S/ 446 329,596 I224V N52H/N57Y/Q100R/H115R/F172S 447 329, 596 N52H/N57Y/Q100R/F172S448 329, 565 N52H/Q100R/H115R/I143T/F172S 449 331, 852N52H/N57Y/Q100P/H115R/F172S 450 333, 853 N52Y/N57Y/Q100P/F172S 451 854,855 E16V/N52H/N57Y/Q100R/V110D/ 452 795, 796 H115R/C198RE16V/N52H/N57Y/Q100R/V110D/ 453 795, 796 H115R/Y152C/K156M/F172S/ C198RN52S/E90A/H115R 454 339, 597 N30D/K42E/N52S/H115R 455 856, 598N30D/K42E/N52S/H115R/C198R/ 456 856, 598 R221IN30D/K42E/N52S/H115R/C198R 457 856, 598 N30D/K42E/N52S/H115R/F172S/ 458856, 598 N194D N52S/H115R/F120S/I143V/C198R 459 197, 857N52S/H115R/F172S/C198R 460 197, 853 N52H/N57Y/Q100P/C198R 461 333, 570N52H/N57Y/Q100P/H115R/F172S/ 462 333, 599 C198RN52H/N57Y/Q100P/F172S/C198R 463 333, 570 N52H/N57Y/Q100P/H115R 464 333,599 N52H/N57Y/Q100P/H115R/C198R 465 333, 599 N52H/Q100R/C198R 466 331,567 N52H/Q100R/H115R/F172S 467 331, 852 N52H/Q100R/H115X/F172S/C198R 468331, 848 N52H/Q100R/H115R/F172S/C198R 469 331, 852N52H/N57Y/Q100R/F172S/C198R 470 329, 565 N52A/N57F/Q100S 638 686, 734N52A/N57H/Q100S 639 687, 735 N52A/N57Y/Q100A 640 688, 736N52D/N57A/Q100A 641 689, 737 N52D/Q100S 642 690, 738 N52G/Q100A 643 691,739 N52H/Q100A 644 692, 740 N52M/N57H/Q100S 645 693, 741 N52M/N57W/Q100P646 694, 742 N52Q/N57F 647 695, 743 N52Q/N57S/Q100A 648 696, 744N52R/N57L/Q100A 649 697, 745 N52R/N57Y/Q100P 650 698, 746N52R/N57Y/Q100S 651 699, 747 N52S/N57A/Q100A 652 700, 748N52S/N57H/Q100E 653 701, 749 N52S/N57L/Q100S 654 702, 750N52S/N57M/Q100S 655 703, 751 N52S/N57Y/Q100S 656 704, 752N52S/N57Y/Q100M 657 705, 753 N52S/N57Y/Q100V 658 706, 754N52T/N57H/Q100S 659 707, 755 N52T/N57H/Q100A 660 708, 756N52T/N57Y/Q100A 661 709, 757 N52V/N57L/Q100A 662 710, 758N52H/N57Y/Q100K 663 711, 759 N52K/N57Y/Q100R 664 712, 760N52L/N57H/Q100R 665 713, 761 N52R/N57F/Q100N 666 714, 762N52R/N57F/Q100P 667 715, 763 N52R/N57F/Q100R 668 716, 764N52R/N57F/Q100T 669 717, 765 N52R/N57H/Q100K 670 718, 766N52R/N57L/Q100S 671 719, 767 N52R/N57W/Q100K 672 720, 768 N52R/N57W 673721, 769 N52R/N57Y/Q100R 674 722, 770 N52C/N57E/Q100S 675 723, 771N52G/N57P/Q100D 676 724, 772 N52G/N57V/Q100G 677 725, 773 N52G/N57V 678726, 774 N52L/N57V 679 727, 775 N52P/N57P 680 728, 776 N52P/N57S/Q100G681 729, 777 N52S/N57L/Q100G 682 730, 778 N52T/N57K/Q100P 683 731, 779N52V/N57T/Q100L 684 732, 780 N57Q/Q100P 685 733, 781 S54F/V193A 905 906,907 R26S/N52H/N57Y/V110D/T137A/ 908 909, 910 C198R

In some embodiments, the variant ICOSL polypeptide exhibits increasedaffinity for the ectodomain of CD28 compared to the reference (e.g.,unmodified) or wild-type ICOSL polypeptide, such as comprising thesequence set forth in SEQ ID NO: 32, 196, or 545. In some embodiments,the ICOSL polypeptide exhibits increased affinity for the ectodomain ofICOS compared to the reference (e.g., unmodified) or wild-type ICOSL,such as comprising the sequence set forth in SEQ ID NO: 32, 196, or 545.In some embodiments, the ICOSL polypeptide exhibits increased affinityfor the ectodomain of CD28 and the ectodomain of ICOS compared to thereference (e.g., unmodified) or wild-type ICOSL, such as comprising thesequence set forth in SEQ ID NO: 32, 196, or 545.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution corresponding to position(s) 52, 54or 57. In some embodiments, the variant ICOSL polypeptide has one ormore amino acid modification, e.g. substitution selected from N52H,N52D, N52Q, N52S, N52Y, N52K, S54A, S54P, or N57Y or a conservativeamino acid modification, e.g. substitution thereof. In some embodiments,the variant ICOSL polypeptide has one or more amino acid modification,e.g. substitution selected from N52H, N52D, N52S, N52K or N57Y or aconservative amino acid modification, e.g. substitution thereof.

In some embodiments, the variant ICOSL polypeptide can contain one ormore further amino acid modification, e.g. substitution in addition toan amino acid modification, e.g. substitution at a positioncorresponding to position 52, 54 or 57. In some embodiments, the one ormore further amino acid modification, e.g. substitution is at a positioncorresponding to 10, 11, 13, 16, 18, 20, 25, 27, 30, 37, 42, 43, 47, 52,54, 57, 61, 62, 67, 71, 72, 74, 75, 77, 78, 80, 84, 89, 90, 92, 93, 94,96, 97, 98, 99, 100, 102, 103, 107, 109, 110, 113, 115, 116, 117, 119,120, 121, 122, 126, 129, 130, 132, 133, 135, 138, 139, 140, 142, 143,144, 146, 151, 152, 153, 154, 155, 156, 158, 161, 166, 168, 172, 173,175, 190, 192, 193, 194, 198, 201, 203, 207, 208, 210, 212, 217, 218,220, 221, 224, 225 or 227. In some embodiments, the one or more furtheramino acid modification, e.g. substitution is at a positioncorresponding to 10, 11, 13, 16, 18, 20, 25, 26, 27, 30, 33, 37, 38, 42,43, 47, 52, 54, 57, 61, 62, 67, 71, 72, 74, 75, 77, 78, 80, 84, 89, 90,92, 93, 94, 96, 97, 98, 99, 100, 102, 103, 107, 109, 110, 111, 113, 115,116, 117, 119, 120, 121, 122, 126, 129, 130, 132, 133, 135, 137, 138,139, 140, 142, 143, 144, 146, 151, 152, 153, 154, 155, 156, 158, 161,164, 166, 168, 172, 173, 175, 190, 192, 193, 194, 198, 201, 203, 207,208, 210, 212, 217, 218, 220, 221, 224, 225, or 227 with reference toSEQ ID NO:32.

In some embodiments, the variant ICOSL contains one or more furtheramino acid modification, e.g. substitution selected from M10V, M10I,V11E, S13G, E16V, S18R, A20V, S25G, F27S, F27C, N30D, Y33del, Q37R,K42E, T43A, Y47H, N52H, N52D, N52S, N52Y, N52K, N52Q, S54A, S54P, N57D,N57Y, R61S, R61C, Y62F, L67P, A71T, G72R, L74Q, R75Q, D77G, F78L, L80P,N84Q, D89G, E90A, K92R, F93L, H94E, H94D, L96F, L96I, V97A, L98F, S99G,Q100R, Q100K, Q100P, L102R, G103E, V107A, V107I, S109G, S109N, V110D,V1100N, V110A, E111del, T113E, H115R, H115Q, V116A, A117T, N119Q, F120I,F120S, S121G, V122A, V122M, S126T, S126R, H129P, S130G, S132F, Q133H,E135K, F138L, T139S, C140del, S142F, I143V, I143T, N144D, Y146C, V151A,Y152C, Y152H, W153R, I154F, K156M, D158G, L161P, L161M, L166Q, N168Q,F172S, L173S, M175T, T190A, T190S, S192G, V193M, N194D, C198R, N201S,L203P, L203F, N207Q, L208P, V210A, S212G, D217V, I218T, I218N, E220G,R221G, R221I, I224V, T225A, N227K, or a conservative amino acidsubstitution thereof. In some embodiments, the variant ICOSL containsone or more further amino acid modification, e.g. substitution selectedfrom M10V, M10I, V11E, S13G, E16V, S18R, A20T, A20V, S25G, R26S, F27C,F27S, N30D, Y33del, Q37R, T38P, K42E, T43A, Y47H, N52A, N52C, N52D,N52G, N52H, N52K, N52L, N52M, N52P, N52Q, N52R, N52S, N52T, N52V, N52Y,S54A, S54F, S54P, N57A, N57D, N57E, N57F, N57H, N57K, N57L, N57M, N57P,N57Q, N57S, N57T, N57V, N57W, N57Y, R61C, R61S, Y62F, L67P, A71T, G72R,L74Q, R75Q, D77G, F78L, L80P, N84Q, D89G, E90A, K92R, F93L, H94D, H94E,L96F, L96I, V97A, L98F, S99G, Q100A, Q100D, Q100E, Q100G, Q100K, Q100L,Q100M, Q100N, Q100P, Q100R, Q100S, Q100T, Q100V, L102R, G103E, V107A,V107I, S109G, S109N, V110A, V110D, V110N, E111del, T113E, H115Q, H115R,V116A, A117T, N119Q, F120I, F120S, S121G, V122A, V122M, S126R, S126T,H129P, S130G, S132F, Q133H, E135K, T137A, F138L, T139S, C140del, C140D,S142F, I143T, I143V, N144D, Y146C, V151A, Y152C, Y152H, W153R, I154F,N155H, N155Q, K156M, D158G, L161M, L161P, Q164L, L166Q, N168Q, F172S,L173S, M175T, T190A, T190S, S192G, V193A, V193M, N194D, C198R, N201S,L203F, L203P, N207Q, L208P, V210A, S212G, D217G, D217V, I218N, I218T,E220G, R221G, R221I, R221K, I224V, T225A, T225S, N227K, or aconservative amino acid substitution thereof.

In some embodiments of any one of the variant ICOSL polypeptidesdescribed above, the variant ICOSL polypeptide further comprises one ormore amino acid deletions corresponding to positions 140 of SEQ ID NO:32.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution selected fromN52Y/N57Y/F138L/L203P, N52H/N57Y/Q100P, N52S/Y146C/Y152C, N52H/C198R,N52H/C140del/T225A, N52H/C198R/T225A, N52H/K92R, N57Y/Q100P, N52S/C198R,N52Y/N57Y/Y152C, N52Y/N57Y/H129P/C198R, N52H/L161P/C198R, N52S/T113E,N52S/S54P, N52K/L208P, N52S/Y152H, N52H/I143T, N52S/R75Q/L203P,N52S/D158G, N52D/Q133H, N52H/N57Y/Q100R/V110D/C198R/S212G,N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/S54P, T38P/N52S/N57D,N52H/C140del/T225A, N52H/F78L/Q100R/C198R, N52H/N57Y/R75Q/Q100P/V110D,N52H/N57Y/L74Q/V110D/S192G, N52H/S121G/C198R, N52S/F120S/N227K,N52S/A71T/A117T/T190A/C198R, T43A/N52H/N57Y/L74Q/D89G/V110D/F172S,N52H/N57Y/Q100R/V110D/S132F/M175T,N52H/N57Y/Q100R/V107I/V110D/I154F/C198R/R221G, N52Q/N207Q, N52Q/N168Q,N52Q/N84Q, N52Q/N9Q, N52Q/N4Q/N68Q, N52Q/N119Q, N52Q/N84Q/N168Q,N52Q/N84Q/N207Q, N52Q/N119Q/N155Q, N52H/N84Q/N119Q, N52H/N84Q,N52H/N84Q/N168Q, N52H/N84Q/N207Q, N52H/N84Q/N168Q/N207Q,N52Q/N84Q/N155Q, N52Q/N84Q/N168Q, N52Q/N84Q/N155Q/N168Q,N52Q/N84Q/N119Q/N168Q, N52Q/N84Q/N119Q/N207Q, N52Q/N84Q/N119Q/N155Q,N52Q/N84Q/N119Q/N155Q/N207Q, N52Y/F138L/L203P, N57Y/Q100R/C198R,N57Y/F138L/L203P, N52H/N57Y/Q100R/H115R/C198R,N52H/N57Y/Q100R/F172S/C198R, N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R,N52H/N57Y/Q100R/L102R/H115R/F172S/C198R, N52H/V122A/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/N194D, N52H/N57Y/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/H115R, N52H/N57Y/Q100R/H115R,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/F172S, N52H/Q100R/H115R/I143T/F172S,N52H/N57Y/Q100P/H115R/F172S, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/E90A/H115R, N30D/K42E/N52S/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N52S/H115R/F120S/I143V/C198R, N52S/H115R/F172S/C198R,N52H/N57Y/Q100P/C198R, N52H/N57Y/Q100P H115R/F172S/C198R,N52H/N57Y/Q100P/F172S/C198R, N52H/N57Y/Q100P/H115R,N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R, N52H/Q100R/H115R/F172S,N52H/Q100R/F172S/C198R, N52H/Q100R/H115R/F172S/C198R, orN52H/N57Y/Q100R/F172S/C198R.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution in an reference ICOSL or specificbinding fragment there of corresponding to position(s) 52, 57, or 100with reference to numbering of SEQ ID NO:32. In some embodiments, thevariant ICOSL polypeptide has one or more amino acid modification, e.g.substitution selected from N52A, N52C, N52D, N52G, N52H, N52L, N52K,N52M, N52P, N52Q, N52R, N52S, N52T, N52V, N52Y, N57A, N57E, N57F, N57H,N57K, N57L, N57M, N57P, N57Q, N57S, N57T, N57V, N57W, N57Y, Q100A,Q100D, Q100E, Q100G, Q100K, Q100L, Q100M, Q100N, Q100R, Q100P, Q100S,Q100T, or Q100V. In some embodiments, the one or more amino acidmodification, e.g. substitution is N52Y/N57Y/F138L/L203P,N52H/N57Y/Q100P, N52S/Y146C/Y152C, N52H/C198R, N52H/C140D/T225A,N52H/C198R/T225A, N52H/K92R, N52H/S99G, N57Y/Q100P, N52S/S130G/Y152C,N52S/Y152C, N52S/C198R, N52Y/N57Y/Y152C, N52Y/N57Y/H129P/C198R,N52H/L161P/C198R, N52S/T113E, N52D/S54P, N52K/L208P, N52S/Y152H,N52D/V151A, N52H/I143T, N52S/L80P, N52S/R75Q/L203P, N52S/D158G,N52D/Q133H, N52S/N57Y/H94D/L96F/L98F/Q100R,N52S/N57Y/H94D/L96F/L98F/Q100R/G103E/F120S, N52H/F78L/Q100R,N52H/N57Y/Q100R/V110D, N52H/N57Y/R75Q/Q100R/V110D, N52H/N57Y/Q100R,N52H/N57Y/L74Q/Q100R/V10D, N52H/Q100R, N52H/S121G,A20V/N52H/N57Y/Q100R/S109G, N52H/N57Y/R61S/Q100R/V110D/L173S,N52H/N57Y/Q100R/V122A, N52H/N57Y/Q100R/F172S, N52H/N57Y, N52S/F120S,N52S/V97A, N52S/G72R, N52S/A71T/A117T, N52S/E220G,Y47H/N52S/V107A/F120S, N52H/N57Y/Q100R/V110D/S132F/M175T,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R,Q37R/N52H/N57Y/Q100R/V110N/S142F/C198R/D217V/R221G,N52H/N57Y/Q100R/V110D/C198R,N52H/N57Y/Q100R/V110D/V116A/L161M/F172S/S192G/C198R,F27S/N52H/N57Y/V110N, N52S/H94E/L96I/S109N/L166Q,S18R/N52S/F93L/I143V/R221G, A20T/N52D/Y146C/Q164L,V11E/N30D/N52H/N57Y/H94E/L96I/L98F/N194D/V210A/I218T,N52S/H94E/L96I/V122M, N52H/N57Y/H94E/L96I/F120I/S126T/W153R/I218N,M10V/S18R/N30D/N52S/S126R/T139S/L203F, S25G/N30D/N52S/F120S/N227K,N30D/N52S/L67P/Q100K/D217G/R221K/T225S,N52H/N57Y/Q100R/V110D/A117T/T190S/C198R,N52H/N57Y/Q100R/V110D/F172S/C198R,S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173S/C198R,N52H/N57Y/V110A/C198R/R221I,M10I/S13G/N52H/N57Y/D77G/V110A/H129P/I143V/F172S/V193M, C198R,N52H/N57Y/R61C/Y62F/Q100R/V110N/F120S/C198R,N52H/N57Y/Q100R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/N144D/F172S/C198R, N52S/H94E/L98F/Q100R,N52S/E90A, N30D/K42E/N52S, N52S/F120S/I143V/I224V,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/S54P, T38P/N52S/N57D,N52H/C140del/T225A, N52H/F78L/Q100R/C198R, N52H/N57Y/R75Q/Q100P/V110D,N52H/N57Y/L74Q/V110D/S192G, N52H/S121G/C198R, N52S/F120S/N227K,N52S/A71T/A117T/T190A/C198R, T43A/N52H/N57Y/L74Q/D89G/V110D/F172S,N52H/N57Y/Q100R/V110D/S132F/M175T, N52D,N52H/N57Y/Q100R/V107I/V110D/I154F/C198R/R221G, N52Q/N207Q, N168Q/N207Q,N52Q/N168Q, N52Q/N84Q, N52Q/N119Q, N52Q/N84Q/N168Q, N52Q/N84Q/N207Q,N52Q/N119Q/N155Q, N52H/N84Q/N119Q, N52H/N84Q, N52H/N84Q/N168Q/N207Q,N52Q/N84Q/N155Q/N168Q, N52Q/N84Q/N119Q/N168Q, N52Q/N84Q/N119Q/N207Q,N52Q/N84Q/N119Q/N155Q, N52Q/N84Q/N119Q/N155Q/N207Q, N52Y/F138L/L203P,N57Y/Q100R/C198R, N57Y/F138L/L203P, Q100R/F138L,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R,N52H/N57Y/Q100R/L102R/H115R/F172S/C198R, N52H/V122A/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/N194D, N52H/N57Y/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/H115R, N52H/N57Y/Q100R/H115R,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/F172S, N52H/Q100R/H115R/I143T/F172S,N52H/N57Y/Q100P/H115R/F172S, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/E90A/H115R, N30D/K42E N52S/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N52S/H115R/F120S/I143V/C198R, N52S/H115R/F172S/C198R,N52H/N57Y/Q100P/C198R, N52H/N57Y/Q100P H115R/F172S/C198R,N52H/N57Y/Q100P/F172S/C198R, N52H/N57Y/Q100P/H115R,N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R, N52H/Q100R/H115R/F172S,N52H/Q100R/F172S/C198R, N52H/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/F172S/C198R, N52A/N57F/Q100S, N52A/N57H/Q100S,N52A/N57Y/Q100A, N52D/N57A/Q100A, N52D/Q100S, N52G/Q100A, N52H/Q100A,N52M/N57H/Q100S, N52M/N57W/Q100P, N52Q/N57F, N52Q/N57S/Q100A,N52R/N57L/Q100A, N52R/N57Y/Q100P, N52R/N57Y/Q100S, N52S/N57A/Q100A,N52S/N57H/Q100E, N52S/N57L/Q100S, N52S/N57M/Q100S, N52S/N57Y/Q100S,N52S/N57Y/Q100M, N52S/N57Y/Q100V, N52T/N57H/Q100S, N52T/N57H/Q100A,N52T/N57Y/Q100A, N52V/N57L/Q100A, N52H/N57Y/Q100K, N52K/N57Y/Q100R,N52L/N57H/Q100R, N52R/N57F/Q100N, N52R/N57F/Q100P, N52R/N57F/Q100R,N52R/N57F/Q100T, N52R/N57H/Q100K, N52R/N57L/Q100S, N52R/N57W/Q100K,N52R/N57W, N52R/N57Y/Q100R, N52C/N57E/Q100S, N52G/N57P/Q100D,N52G/N57V/Q100G, N52G/N57V, N52L/N57V, N52P/N57P, N52P/N57S/Q100G,N52S/N57L/Q100G, N52T/N57K/Q100P, N52V/N57T/Q100L, or N57Q/Q100P.

In some embodiments, the one or more amino acid modifications areselected from among N52Y/N57Y/F138L/L203P, N52H/N57Y/Q100P,N52S/Y146C/Y152C, N52H/C198R, N52H/C140D/T225A, N52H/C198R/T225A,N52H/K92R, N52H/S99G, N57Y/Q100P, N52S/S130G/Y152C, N52S/Y152C,N52S/C198R, N52Y/N57Y/Y152C, N52Y/N57Y/H129P/C198R, N52H/L161P/C198R,N52S/T113E, N52D/S54P, N52K/L208P, N52S/Y152H, N52D/V151A, N52H/I143T,N52S/L80P, F120S/Y152H/N201S, N52S/R75Q/L203P, N52S/D158G, N52D/Q133H,N52S/N57Y/H94D/L96F/L98F/Q100R,N52S/N57Y/H94D/L96F/L98F/Q100R/G103E/F120S, N52S/G103E, N52H/F78L/Q100R,N52H/N57Y/Q100R/V110D, N52H/N57Y/R75Q/Q100R/V110D, N52H/N57Y/Q100R,N52H/N57Y/L74Q/Q100R/V110D, N52H/Q100R, N52H/S121G,A20V/N52H/N57Y/Q100R/S109G, N52H/N57Y/R61S/Q100R/V110D/L173S,N52H/N57Y/Q100R/V122A, N52H/N57Y/Q100R/F172S, N52H/N57Y, N52S/F120S,N52S/V97A, N52S/G72R, N52S/A71T/A117T, N52S/E220G,Y47H/N52S/V107A/F120S, N52H/N57Y/Q100R/V110D/S132F/M175T,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R,Q37R/N52H/N57Y/Q100R/V110N/S142F/C198R/D217V/R221G,N52H/N57Y/Q100R/V110D/C198R,N52H/N57Y/Q100R/V110D/V116A/L161M/F172S/S192G/C198R,F27S/N52H/N57Y/V110N, N52S/H94E/L96I/S109N/L166Q,S18R/N52S/F93L/I143V/R221G, A20T/N52D/Y146C/Q164L,V11E/N30D/N52H/N57Y/H94E/L96I/L98F/N194D/V210A/I218T,N52S/H94E/L96I/V122M, N52H/N57Y/H94E/L96I/F120I/S126T/W153R/I218N,M10V/S18R/N30D/N52S/S126R/T139S/L203F, S25G/N30D/N52S/F120S/N227K,N30D/N52S/L67P/Q100K/D217G/R221K/T225S,N52H/N57Y/Q100R/V110D/A117T/T190S/C198R,N52H/N57Y/Q100R/V110D/F172S/C198R,S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173S/C198R,N52H/N57Y/V110A/C198R/R221I,M10I/S13G/N52H/N57Y/D77G/V110A/H129P/I1143V/F172S/V193M, C198R,N52H/N57Y/R61C/Y62F/Q100R/V110N/F120S/C198R,N52H/N57Y/Q100R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/N144D/F172S/C198R, N52S/H94E/L98F/Q100R,N52S/E90A, N30D/K42E/N52S, N52S/F120S/I143V/I224V,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/S54P, T38P/N52S/N57D,N52H/C140del/T225A, N52H/F78L/Q100R/C198R, N52H/N57Y/R75Q/Q100P/V110D,N52H/N57Y/L74Q/V110D/S192G, N52H/S121G/C198R, N52S/F120S/N227K,N52S/A71T/A117T/T190A/C198R, T43A/N52H/N57Y/L74Q/D89G/V110D/F172S,N52H/N57Y/Q100R/V110D/S132F/M175T,N52H/N57Y/Q100R/V107I/V110D/I154F/C198R/R221G, N52Q/N207Q, N52Q/N168Q,N52Q/N84Q, N52Q/N119Q, N52Q/N84Q/N168Q, N52Q/N84Q/N207Q,N52Q/N119Q/N155Q, N52H/N84Q/N119Q, N52H/N84Q, N52H/N84Q/N168Q/N207Q,N52Q/N84Q/N155Q/N168Q, N52Q/N84Q/N119Q/N168Q, N52Q/N84Q/N119Q/N207Q,N52Q/N84Q/N119Q/N155Q, N52Q/N84Q/N119Q/N155Q/N207Q, N52Y/F138L/L203P,N57Y/Q100R/C198R, N57Y/F138L/L203P, Q100R/F138L,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R,N52H/N57Y/Q100R/L102R/H115R/F172S/C198R, N52H/V122A/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/N194D, N52H/N57Y/H115R/F172S/C198R,N52H/N57Y/H115R, N52H/N57Y/Q100R/H115R,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/F172S, N52H/Q100R/H115R/I143T/F172S,N52H/N57Y/Q100P/H115R/F172S, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/E90A/H115R, N30D/K42E N52S/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N52S/H115R/F120S/I143V/C198R, N52S/H115R/F172S/C198R,N52H/N57Y/Q100P/C198R, N52H/N57Y/Q100P/H115R/F172S/C198R,N52H/N57Y/Q100P/F172S/C198R, N52H/N57Y/Q100P/H115R,N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R, N52H/Q100R/H115R/F172S,N52H/Q100R/F172S/C198R, N52H/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/F172S/C198R, N52A/N57F/Q100S, N52A/N57H/Q100S,N52A/N57Y/Q100A, N52D/N57A/Q100A, N52D/Q100S, N52G/Q100A, N52H/Q100A,N52M/N57H/Q100S, N52M/N57W/Q100P, N52Q/N57F, N52Q/N57S/Q100A,N52R/N57L/Q100A, N52R/N57Y/Q100P, N52R/N57Y/Q100S, N52S/N57A/Q100A,N52S/N57H/Q100E, N52S/N57L/Q100S, N52S/N57M/Q100S, N52S/N57Y/Q100S,N52S/N57Y/Q100M, N52S/N57Y/Q100V, N52T/N57H/Q100S, N52T/N57H/Q100A,N52T/N57Y/Q100A, N52V/N57L/Q100A, N52H/N57Y/Q100K, N52K/N57Y/Q100R,N52L/N57H/Q100R, N52R/N57F/Q100N, N52R/N57F/Q100P, N52R/N57F/Q100R,N52R/N57F/Q100T, N52R/N57H/Q100K, N52R/N57L/Q100S, N52R/N57W/Q100K,N52R/N57W, N52R/N57Y/Q100R, N52C/N57E/Q100S, N52G/N57P/Q100D,N52G/N57V/Q100G, N52G/N57V, N52L/N57V, N52P/N57P, N52P/N57S/Q100G,N52S/N57L/Q100G, N52T/N57K/Q100P, N52V/N57T/Q100L, N57Q/Q100P, orR26S/N52H/N57Y/V110D/T137A/C198R.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution selected from N52A, N52C, N52D,N52G, N52K, N52L, N52M, N52R, N52T, N52V, N57A, N57E, N57F, N57H, N57K,N57L, N57M, N57P, N57Q, N57S, N57T, N57V, N57W, Q100A, Q100D, Q100G,Q100L, Q100M, Q100N, Q100R, Q100S, Q100T or Q100V. with reference to SEQID NO:32. In some embodiments, the one or more amino acid modificationsare selected from among N52A/N57F/Q100S, N52A/N57H/Q100S,N52A/N57Y/Q100A, N52D/N57A/Q100A, N52D/Q100S, N52G/Q100A, N52H/Q100A,N52M/N57H/Q100S, N52M/N57W/Q100P, N52Q/N57F, N52Q/N57S/Q100A,N52R/N57L/Q100A, N52R/N57Y/Q100P, N52R/N57Y/Q100S, N52S/N57A/Q100A,N52S/N57H/Q100E, N52S/N57L/Q100S, N52S/N57M/Q100S, N52S/N57Y/Q100S,N52S/N57Y/Q100M, N52S/N57Y/Q100V, N52T/N57H/Q100S, N52T/N57H/Q100A,N52T/N57Y/Q100A, N52V/N57L/Q100A, N52H/N57Y/Q100K, N52K/N57Y/Q100R,N52L/N57H/Q100R, N52R/N57F/Q100N, N52R/N57F/Q100P, N52R/N57F/Q100R,N52R/N57F/Q100T, N52R/N57H/Q100K, N52R/N57L/Q100S, N52R/N57W/Q100K,N52R/N57W, N52R/N57Y/Q100R, N52C/N57E/Q100S, N52G/N57P/Q100D,N52G/N57V/Q100G, N52G/N57V, N52L/N57V, N52P/N57P, N52P/N57S/Q100G,N52S/N57L/Q100G, N52T/N57K/Q100P, N52V/N57T/Q100L or N57Q/Q100P.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution selected fromN52H/N57Y/Q100R/C198R, N52H/N57Y/Q100R/V122A, N52H/N57Y/Q100R/F172S,N52Y/N57Y/F138L/L203P,V11E/N30D/N52H/N57Y/H94E/L96I/L98F/N194D/V210A/I218T,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52H/N57Y/Q100R, N52H/Q100R,N52H/N57Y/Q100R/V110D/C198R/S212G,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/V152C/K156M/C198R, N30D/K42E/N52S,N52S/F120S/I143V/I224V, N52S/E90A, N52H/N57Y/V110A/C198R/R221I,N52H/N57Y/Q100P, or N52S/N194D.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution selected fromN52H/N57Y/Q100R/F172S, N52H/Q100R, or N52H/N57Y/Q100R/C198R. In someembodiments, the variant ICOSL polypeptide has one or more amino acidmodification, e.g. substitution selected fromE16V/N52H/N57Y/Q100R/V1100D/H115R/Y152C/K156M/C198R, N52H/N57Y/Q100R,and N52H/N57Y/Q100P.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution selected fromN52H/N57Y/F138L/L203P, N52H/N57Y/Q100P, N52H/K92R, N52H/C140del/T225A,N52H/C198R/T225A, N52H/K92R, N57Y/Q100P, N52Y/N57Y/H129P/C198R,N52H/L161P/C198R, N52K/L208P or N52H/I143T.

In some embodiments, the one or more amino acid modifications areselected from among F120S/Y152H/N201S, E111del, Y33del, N168Q/N207Q,N84Q/N207Q, N155Q/N207Q, N119Q/N168Q, N119Q/N207Q, N119Q/N155Q,N84Q/N119Q, N84Q/N155Q/N168Q, N84Q/N168Q/N207Q, N84Q/N155H/N207Q,N155Q/N168Q/N207Q, N119Q N155Q/N168Q, N119Q/N168Q/N207Q,N84Q/N119Q/N207Q, N119Q/N155H/N207Q, N84Q/N119Q/N155Q,N84Q/N119Q/N155Q/N168Q, N84Q/N155Q/N168Q/N207Q, N84Q/N119Q/N155Q/N207Q,N84Q/N119Q/N155Q/N168Q/N207Q or F138L/L203P.

In some embodiments, the variant ICOSL polypeptide exhibits increasedbinding affinity for binding one of the ectodomains of CD28 or ICOS andexhibits decreased binding affinity for binding to the other of theectodomains of CD28 or ICOS compared to the reference (e.g., unmodified)or wild-type ICOSL polypeptide, such as comprising the sequence setforth in SEQ ID NO: 32, 196, or 545.

In some embodiments, the variant ICOSL polypeptide exhibits increasedbinding affinity for ICOS. In some embodiments, the one or more aminoacid substitution is at a position corresponding to 16, 30, 42, 52, 54,57, 75, 90, 92, 100, 102, 110, 113, 115, 120, 122, 133, 138, 143, 146,152, 156, 158, 172, 194, 198, 203, 208, 221, 224, or 225. In someembodiments, the variant ICOSL contains one or more amino acidsubstitutions selected from C198R, D158G, E16V, E90A, F120S, F138L,F172S, H115R, H115X, I143T, I143V, I224V, K156M, K42E, K92R, L102R,L203P, L208P, N194D, N30D, N52A, N52D, N52G, N52H, N52K, N52L, N52M,N52Q, N52R, N52S, N52T, N52Y, N57F, N57H, N57K, N57L, N57M, N57P, N57S,N57V, N57W, N57Y, Q100A, Q100D, Q100E, Q100K, Q100M, Q100P, Q100P,Q100R, Q100S, Q100T, Q133H, R221I, R75Q, S54A, S54P, T113E, T225A,V110D, V122A, Y146C, Y152C, or a conservative amino acid substitutionthereof. In some embodiments, the variant ICOSL polypeptide has one ormore amino acid substitutions selected from N52S, N52H, N52D,N52H/N57Y/Q100P, N52S/Y146C/Y152C, N52H/C198R, N52H/C198R/T225A,N52H/K92R, N57Y, N52S/C198R, N52S/T113E, S54A, N52D/S54P, N52K/L208P,N52H/I143T, N52S/R75Q/L203P, N52S/D158G, N52D/Q133H,N52H/N57Y/Q100R/V122A, N52H/N57Y/Q100R/F172S, N52H/N57Y/Q100R,N52S/N194D, N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/E90A,N52S/F120S/I143V/I224V, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/H115R/F120S/I143V/C198R, N52H/N57Y/Q100P/C198R,N52H/N57Y/Q100P/H115R/F172S/C198R, N52H/N57Y/Q100P/F172S/C198R,N52H/N57Y/Q100P/H115R, N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R,N52H/Q100R/H115X/F172S/C198R, N52H/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/H115R/F172S/C198R, Q100R, N52Y/F138L/L203P,N57Y/Q100R/C198R, N57Y/F138L/L203P, N57Y/Q100P, Q100R/F138L,N52H/N57Y/Q100R/H115R, N52H/N57Y/Q100R/F172S,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R, N52H/N57Y/Q100R/L102R,H115R/F172S/C198R, N52H/N57Y/Q100R/H115R F172S/N194D,N52H/N57Y/H115R/F172S/C198R, N52H/N57Y/Q100R/H115R/C198R,N52H/N57Y/H115R, N52H/Q100R/H115R/I143T F172S,N52H/N57Y/Q100P/H115R/F172S, E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,N52S/E90A/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N30D/K42E/N52S/H115R, N52S/E90A/H115R, N30D/K42E/N52S/H115R,N52A/N57H/Q100S, N52A/N57Y/Q100A, N52D/Q100S, N52G/Q100A,N52M/N57H/Q100S, N52M/N57W/Q100P, N52Q/N57S/Q100A, N52R/N57L/Q100A,N52S/N57H/Q100E, N52S/N57L/Q100S, N52S/N57M/Q100S, N52S/N57Y/Q100M,N52T/N57H/Q100S, N52R/N57F/Q100P, N52R/N57F/Q100T, N52R/N57W/Q100K,N52R/N57W, N52G/N57P/Q100D, N52G/N57V/Q100G, N52G/N57V, N52L/N57V,N52S/N57L/Q100G or N52T/N57K/Q100P.

In some embodiments, the variant ICOSL polypeptide exhibits increasedbinding affinity for ICOS and exhibits decreased binding affinity forCD28. In some embodiments, the one or more further amino acidsubstitution is at a position corresponding to 52, 57, 80 100, 130, 152,161 or 198. In some embodiments, the variant ICOSL contains one or moreamino acid substitutions selected from N52S, N52H, N52Y, N52H, N57Y,L80P, Q100P Q100R, Q100K, V110D, S130G, Y152C, L161P, L161M, C198R,R221G, or a conservative amino acid substitution thereof. In someembodiments, the variant ICOSL polypeptide has one or more amino acidsubstitutions selected from N57Y/Q100P, N52S/S130G/Y152C, N52S/Y152C,N52Y/N57Y/Y152C, N52H/L161P/C198R, N52H/L161P/C198R, N52S/L80P,A20V/N52H/N57Y/Q100R/S109G, N52H/N57Y/R61S/Q100R/V110D/L173S,N52H/N57Y/Q100R/V107I/V110D/S132F/I154F/C198R/R221G,Q37R/N52H/N57Y/Q100R/V110N/S142F/C198R/D217V/R221G,N52H/N57Y/Q100R/V110D/C198R, F27S/N52H/N57Y/V110N,S18R/N52S/F93L/I143V/R221G, A20T/N52D/Y146C/Q164L,N52H/N57Y/H94E/L96/F120I/S126T/W153R/I218N,N52H/N57Y/Q100R/V110D/F172S/C198R,S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173 S/C198R,M10I/S13G/N52H/N57Y/D77G/V110A/H129P/I143V/F172S/V193M/C198R.

In some embodiments, the variant ICOSL polypeptide exhibits increasedbinding affinity for CD28. In some embodiments, the one or more aminoacid substitution is at a position corresponding to 10, 11, 13, 16, 18,20, 25, 27, 30, 36, 40, 41, 42, 52, 54, 57, 63, 70, 71, 72, 74, 77, 80,81, 84, 88, 89, 90, 91, 92, 93, 94, 96, 98, 99, 100, 102, 107, 109, 110,113, 114, 115, 117, 118, 119, 120, 121, 122, 126, 127, 129, 130, 132,133, 135, 138, 139, 140, 143, 144, 146, 152, 153, 154, 155, 156, 158,161, 166, 168, 169, 172, 173, 178, 190, 192, 193, 194, 198, 199, 201,203, 207, 208, 209, 212, 218, 221, 224, 225, or 227.

In some embodiments, the variant ICOSL contains one or more amino acidsubstitutions selected from A117T, A20V, A71T, A91G, A91G, AE88D,C140del, C198R, D158G, D77G, D90K, E117G, E135K, E16V, E81A, E88D, E90A,F120I, F120S, F138L, F172S, F27C, F92Y, G72R, H115R, H115X, H129P, H94E,I118V, I127T, I143T, I143V, I154F, 1218N, I218T, I224V, K156M, K169E,K36G, K42E, K89R, K92R, K93R, L102R, L161P, L166Q, L173S, L203F, L203P,L208P, L209P, L40M, L70Q, L70R, L74Q, L80P, L96I, L98F, M10I, M10V,N115Q, N119Q, N122S, N144D, N155X, N168Q, N168X, N178S, N194D, N207Q,N207X, N227K, N25S, N30D, N52A, N52D, N52G, N52H, N52K, N52L, N52M,N52Q, N52R, N52S, N52T, N52V, N52Y, N57A, N57F, N57H, N57L, N57M, N57S,N57V, N57W, N57Y, N63S, N84Q, Q100A, Q100E, Q100G, Q100K, Q100M, Q100N,Q100P, Q100R, Q100S, Q100T, Q100V, Q133H, R221G, R221I, S109G, S109N,S114T, S121G, S126R, S126T, S130G, S132F, S13G, S18R, S192G, S212G,S25G, S54A, S54P, S99G, T113E, T120S, T130A, T139S, T190A, T199S, T225A,T41I, V107I, V110A, V110D, V11E, V122A, V122M, V193M, V210A, W153R,Y146C, Y152C, Y152H, or a conservative amino acid substitution thereof.In some embodiments, the variant ICOSL polypeptide has one or more aminoacid substitutions selected from N52S, N52H, N52D,N52Y/N57Y/F138L/L203P, N52H/N57Y/Q100P, N52S/Y146C/Y152C, N52H/C198R,N52H/C140del/T225A, N52H/C198R/T225A, N52H/K92R, N52H/S99G, N57Y,N57Y/Q100P, N52S/S130G/Y152C, N52S/Y152C, N52S/C198R, N52Y/N57Y/Y152C,N52Y/N57Y/H129P/C198R, N52H/L161P/C198R, N52S/T113E, S54A, N52D/S54P,N52K/L208P, N52S/Y152H, N52H/I143T, N52S/L80P, N52S/D158G, N52D/Q133H,L70Q/A91G/N144D, L70Q/A91G/E117G/I118V/T120S/T130A,L70R/A91G/I118V/T120S/T130A/T199S,L70Q/E81A/A91G/I118V/T120S/I127T/T130A,N63S/L70Q/A91G/S114T/I118V/T120S/T130A, T41I/A91G,E88D/K89R/D90K/A91G/F92Y/K93R/N122S/N178S,E88D/K89R/D90K/A91G/F92Y/K93R, AE88D/K89R/D90K/A91G/F92Y/K93R,K36G/L40M, N52H/N57Y/Q100R/V122A, N52H/N57Y/Q100R/F172S,N52H/N57Y/Q100R, N52S/F120S/N227K, N52S/N194D, N52S/F120S, N52S/G72R,N52S/A71T/A117T/T190A/C198R,N52H/N57Y/Q100R/V107I/V110D/S132F/I154F/C198R/R221G,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R,N52H/N57Y/Q100R/V110D/C198R,V11E/N30D/N52H/N57Y/H94E/L96I/L98F/N194D/V210A/I218T,N52S/H94E/L96I/V122M, N52H/N57Y/H94E/L96I/F120/S126T/W153R/I218N,M10V/S18R/N30D/N52S/S126R/T139S/L203F, S25G/N30D/N52S/F120S/N227K,N52H/N57Y/Q100R/V110D/F172S/C198R,S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173S/C198R,N52H/N57Y/V110A/C198R/R221I,M1I/S13G/N52H/N57Y/D77G/V110A/H129P/I143V/F172S/V193M, C198R,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/N144D/F172S/C198R, N52S/H94E/L98F/Q100R,N52S/E90A, N52S/F120S/I143V/I224V, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/H115R/F120S/I143V/C198R, N52H/N57Y/Q100P/C198R,N52H/N57Y/Q100P/H115R/F172S/C198R, N52H/N57Y/Q100P/F172S/C198R,N52H/N57Y/Q100P/H115R, N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R,N52H/Q100R/H115R/F172S, N52H/Q100R/H115X/F172S/C198R,N52H/Q100R/H115R/F172S/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/H115R/F172S/C198R, N52S/H94E/L96I/S109N/L166Q/,N52H/N57Y/Q100R/C198R, N52H/N57Y/L74Q/V110D/S192G, N52H/Q100R,N52H/S121G/C198R, A20V/N52H/N57Y/Q100R/S109G, N52H/N57Y/Q100P/C198R,N52H/N57Y/Q100R/V110D/C198R/S212G, L70Q/A91G/I118A/T120S/T130A/K169E,Q100R, N52Y/F138L/L203P, N57Y/Q100R/C198R, N57Y/F138L/L203P, N52H, N57Y,N57Y/Q100P, N52H/N57Y/Q100R/H115R, N52H/N57Y/Q100R/F172S,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R, N52H/N57Y/Q100R/L102RH115R/F172S/C198R, N52H/N57Y/Q100R/H115R F172S/N194D,N52H/N57Y/H115R/F172S/C198R, N52H/N57Y/Q100R/H115R/C198R,N52H/N57Y/H115R, N52H/Q100R/H115R/I143T F172S,N52H/N57Y/Q100P/H115R/F172S, E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,N30D/K42E/N52S/H115R/C198R R221I, N52S/E90A/H115R, N30D/K42E/N52S/H115R,N52S/H115R/F172S/C198R, N119Q, N207Q, N52Q/N207X, N168X/N207X,N52Q/N168Q, N84Q/N207Q, N119Q N155X, N52Q/N119Q, N52Q/N84Q/N207Q,N119Q/N155Q/N168Q, N52H/N84Q/N119Q, N52Q/N84Q/N155X/N168X,N52A/N57F/Q100S, N52A/N57H/Q100S, N52A/N57Y/Q100A, N52D/N57A/Q100A,N52D/Q100S, N52G/Q100A, N52H/Q100A, N52M/N57H/Q100S, N52M/N57W/Q100P,N52Q/N57F, N52Q/N57S/Q100A, N52R/N57L/Q100A, N52R/N57Y/Q100P,N52R/N57Y/Q100S, N52S/N57A/Q100A, N52S/N57H/Q100E, N52S/N57L/Q100S,N52S/N57M/Q100S, N52S/N57Y/Q100S, N52S/N57Y/Q100M, N52S/N57Y/Q100V,N52T/N57H/Q100S, N52T/N57H/Q100A, N52T/N57Y/Q100A, N52V/N57L/Q100A,N52H/N57Y/Q100K, N52K/N57Y/Q100R, N52L/N57H/Q100R, N52R/N57F/Q100N,N52R/N57F/Q100P, N52R/N57F/Q100R, N52R/N57F/Q100T, N52R/N57L/Q100S,N52R/N57W/Q100K, N52R/N57W, N52G/N57V, N52L/N57V, N52S/N57L/Q100G, orN52T/N57K/Q100P.

In some embodiments, the variant ICOSL polypeptide exhibits increasedbinding affinity for CD28 and exhibits decreased binding affinity forICOS. In some embodiments, the one or more amino acid substitution is ata position corresponding to 52, 75 or 203. In some embodiments, thevariant ICOSL contains one or more amino acid substitution selected fromN52S, R75Q, L203F, or L203P. In some embodiments, the variant ICOSLpolypeptide has amino acid substitutions N52S/R75Q/L203P.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution in an reference ICOSL or specificbinding fragment there of corresponding to position(s) 16, 30, 42, 52,57, 90, 100, 102, 110, 115, 120, 122, 138, 143, 152, 156, 172, 194, 198,203, 221, or 224 with reference to numbering of SEQ ID NO: 32. In someembodiments, the variant ICOSL polypeptide has one or more amino acidmodification, e.g. substitution selected from E16V, N30D, K42E, N52H,N52Y, N52S, N57Y, E90A, Q100R, Q100P, L102R, V110D, H115R, F120S, V122A,F138L, I143V, I143T, H152C, K156M, F172S, N194D, C198R, L203P, R221I, orI224V. In some embodiments, the variant ICOSL polypeptide has one ormore amino acid modification, e.g. substitution in an reference ICOSL orspecific binding fragment there of corresponding to position(s) 115,172, or 198 with reference to numbering of SEQ ID NO:32. In someembodiments, the variant ICOSL polypeptide has one or more amino acidmodification, e.g. substitution selected from H115R, F172S or C198R. Insome embodiments, the one or more amino acid modification, e.g.substitution is N52H/N57Y/Q100R/H115R/C198R,N52H/N57Y/Q100R/F172S/C198R, N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R,N52H/N57Y/Q100R/L102R/H115R/F172S/C198R, N52H/V122A/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/N194D, N52H/N57Y/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/H115R, N52H/N57Y/Q100R/H115R,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/F172S, N52H/Q100R/H115R/I143T/F172S,N52H/N57Y/Q100P/H115R/F172S, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/E90A/H115R, N30D/K42E N52S/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N52S/H115R/F120S/I143V/C198R, N52S/H115R/F172S/C198R,N52H/N57Y/Q100P/C198R, N52H/N57Y/Q100P H115R/F172S/C198R,N52H/N57Y/Q100P/F172S/C198R, N52H/N57Y/Q100P/H115R,N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R, N52H/Q100R/H115R/F172S,N52H/Q100R/F172S/C198R, N52H/Q100R/H115R/F172S/C198R orN52H/N57Y/Q100R/F172S/C198R. In some embodiments, the variant ICOSLpolypeptides exhibit potentially enhanced protein solubility or enhancedprotein expression (‘solubility mutations’) compared to the reference(e.g., unmodified) or wild-type ICOSL polypeptide.

In some embodiments, the variant ICOSL polypeptide comprises any of theextracellular domain (ECD) sequences set forth in SEQ ID NOS: 435-470.In some embodiments, the variant ICOSL polypeptide comprises apolypeptide sequence that exhibits at least 90% identity, at least 91%identity, at least 92% identity, at least 93% identity, at least 94%identity, at least 95% identity, such as at least 96% identity, 97%identity, 98% identity, or 99% identity to any of the extracellulardomain (ECD) set forth in SEQ ID NOS: 435-470 and contains the aminoacid modification(s), e.g. substitution(s) not present in the reference(e.g., unmodified) or wild-type ICOSL. In some embodiments, the variantICOSL polypeptide comprises a specific binding fragment of any of theextracellular domain (ECD) sequences set forth in SEQ ID NOS: 435-470and contains the amino acid modification(s), e.g. substitution (s) notpresent in the reference (e.g., unmodified) or wild-type ICOSL.

In some embodiments, the variant ICOSL polypeptide exhibits increasedbinding affinity for CD28 and exhibits increased binding affinity forICOS. In some embodiments, the one or more amino acid substitution is ata position corresponding to 16, 30, 42, 52, 54, 57, 90, 92, 100, 102,110, 113, 115, 120, 122, 133, 138, 143, 146, 152, 156, 158, 172, 194,198, 203, 208, 212, 224, or 225. In some embodiments, the variant ICOSLcontains one or more amino acid substitutions selected from C198R,D158G, E16V, E90A, F120S, F138L, F172S, H115R, 1143V, I224V, K156M,K42E, K92R, L102R, L203P, L208P, N194D, N30D, N52A, N52D, N52G, N52H,N52K, N52L, N52M, N52Q, N52R, N52S, N52T, N52Y, N57F, N57H, N57L, N57M,N57S, N57V, N57W, N57Y, Q100A, Q100E, Q100G, Q100K, Q100M, Q100P, Q100R,Q100S, Q133H, S212G, S54A, S54P, T113E, T225A, V110D, V122A, Y146C,Y152C, or a conservative amino acid substitution thereof. In someembodiments, the variant ICOSL polypeptide has one or more amino acidsubstitutions selected from N52A/N57Y/Q100A, N52D/Q100S, N52G/Q100A,N52M/N57H/Q100S, N52M/N57W/Q100P, N52Q/N57S/Q100A, N52R/N57L/Q100A,N52S/N57H/Q100E, N52S/N57L/Q100S, N52S/N57M/Q100S, N52S/N57Y/Q100M,N52T/N57H/Q100S, N52R/N57F/Q100P, N52R/N57F/Q100T, N52R/N57W/Q100K,N52R/N57W, N52G/N57V, N52L/N57V, N52S/N57L/Q100G, N52T/N57K/Q100P, N52S,N52H, N52D, N52Y/N57Y/F138L/L203P, N52H/N57Y/Q100P, N52S/Y146C/Y152C,N52H/C198R, N52H/C198R/T225A, N52H/K92R, N57Y, N52S/C198R, N52S/T113E,S54A, N52D/S54P, N52K/L208P, N52H/I143T, N52S/D158G, N52D/Q133H,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/V122A,N52H/N57Y/Q100R/F172S, N52H/N57Y/Q100R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/E90A,N52S/F120S/I143V/I224V, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/H115R/F120S/I143V/C198R, N52H/N57Y/Q100P/C198R,N52H/N57Y/Q100P/H115R/F172S/C198R, N52H/N57Y/Q100P/F172S/C198R,N52H/N57Y/Q100P/H115R, N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R,N52H/Q100R/H115R/F172S, N52H/Q100R/H115X/F172S/C198R,N52H/Q100R/H115R/F172S/C198R, N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S, N52H/N57Y/Q100R/H115R/F172S/C198R, Q100R,N52Y/F138L/L203P, N57Y/Q100R/C198R, N57Y/F138L/L203P, N52H, N57Y,N57Y/Q100P, Q100R/F138L, N52H/N57Y/Q100R/H115R, N52H/N57Y/Q100R/F172S,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R, N52H/N57Y/Q100R/L102RH115R/F172S/C198R, N52H/N57Y/Q100R/H115R F172S/N194D,N52H/N57Y/H115R/F172S/C198R, N52H/N57Y/Q100R/H115R/C198R,N52H/N57Y/H115R, N52H/Q100R/H115R/I143T/F172S,N52H/N57Y/Q100P/H115R/F172S, E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,N52S/E90A/H115R, N52S/E90A/H115R, N30D/K42E/N52S/H115R.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution in a reference ICOSL or specificbinding fragment there of corresponding to position(s) 52, 57, 100, 138,198, or 203 with reference to numbering of SEQ ID NO:32. In someembodiments, the variant ICOSL polypeptide has one or more amino acidmodification, e.g. substitution selected from N52H, N52Y, N57Y, Q100R,Q100P, F138L, C198R, or L203P. In some embodiments, the one or moreamino acid modification, e.g. substitution is Q100R, F138L/L203P,N52Y/F138L/L203P, N57Y/Q100R/C198R, N57Y/F138L/L203P, N52H, N57Y,N57Y/Q100P, Q100R/F138L, or L203P.

In some embodiments, the variant ICOSL polypeptide comprises any of theextracellular domain (ECD) sequences set forth in SEQ ID NOS: 427-433.In some embodiments, the variant ICOSL polypeptide comprises apolypeptide sequence that exhibits at least 90% identity, at least 91%identity, at least 92% identity, at least 93% identity, at least 94%identity, at least 95% identity, such as at least 96% identity, 97%identity, 98% identity, or 99% identity to any of the extracellulardomain (ECD) set forth in SEQ ID NOS: 427-433 and contains the aminoacid modification(s), e.g. substitution(s) not present in the reference(e.g., unmodified) or wild-type ICOSL. In some embodiments, the variantICOSL polypeptide comprises a specific binding fragment of any of theextracellular domain (ECD) sequences set forth in SEQ ID NOS: 427-433and contains the amino acid modification(s), e.g. substitution (s) notpresent in the reference (e.g., unmodified) or wild-type ICOSL. In someembodiments, the variant ICOSL polypeptide comprises the IgV sequenceset forth in SEQ ID NO: 434. In some embodiments, the variant ICOSLpolypeptide comprises a polypeptide sequence that exhibits at least 90%identity, at least 91% identity, at least 92% identity, at least 93%identity, at least 94% identity, at least 95% identity, such as at least96% identity, 97% identity, 98% identity, or 99% identity to the IgVsequence set forth in SEQ ID NO: 434 and contains the amino acidmodification(s), e.g. substitution(s) not present in the reference(e.g., unmodified) or wild-type ICOSL. In some embodiments, the variantICOSL polypeptide comprises a specific binding fragment of the IgVsequence set forth in SEQ ID NO: 434 and contains the amino acidsubstitution(s) not present in the reference (e.g., unmodified) orwild-type ICOSL.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution in a reference ICOSL or specificbinding fragment there of corresponding to position(s) 52, 84, 91, 119,155, 168, 207 with reference to numbering of SEQ ID NO:32. In someembodiments, the variant ICOSL polypeptide has one or more amino acidmodification, e.g. substitution selected from A91S, N52H, N52Q, N84Q,N119Q, N155H, N155Q, N168Q, N207Q. In some embodiments, the one or moreamino acid modification, e.g. substitution is N84Q, N119Q, N168Q, N207Q,N52Q, N52Q/N207Q, N168Q/N207Q, N52Q/N168Q, N84Q/N207Q, N155Q/N207Q,N119Q/N168Q, N119Q/N207Q, N119Q/N155Q, N52Q/N84Q, N52Q/N119Q,N84Q/N119Q, N52Q/N84Q/N168Q, N52Q/N84Q/N207Q, N84Q/N155Q/N168Q,N84Q/N168Q/N207Q, N84Q/N155H/N207Q, N155Q/N168Q/N207Q, N119QN155Q/N168Q, N119Q/N168Q/N207Q, N84Q/N119Q/N207Q, N119Q/N155H/N27Q,N4Q/N119Q1/N155Q, N52Q/N119Q/N155Q, N52H/N84Q/N119Q, N52H/N84Q,N52H/N84Q/N168Q, N52H/N84Q/N207Q, N52H/N84Q/N168Q/N207Q,N52Q/N84Q/N155Q, N52Q/N84Q/N168Q, N52Q/N84Q/N155Q/N168Q,N52Q/N84Q/N119Q/N168Q, N84Q/N119Q/N155Q/N168Q, N84Q/N155Q/N168Q/N207Q,N84Q/N119Q/N155Q/N207Q, N52Q/N84Q/N119Q/N207Q, N52Q/N84Q/N119Q/N155Q,N52Q/N84Q/N119Q/N155Q/N207Q, N84Q/N119Q/N155Q/N168Q/N207Q orA91S/N119Q/N168Q/N207Q. In some embodiments, the variant ICOSLpolypeptides exhibit potentially reduced glycosylation compared to thereference (e.g., unmodified) or wild-type ICOSL polypeptide.

In some embodiments, the variant ICOSL polypeptide comprises any of theextracellular domain (ECD) sequences set forth in SEQ ID NOS: 387-424,427-433, 435-470. In some embodiments, the variant ICOSL polypeptidecomprises a polypeptide sequence that exhibits at least 90% identity, atleast 91% identity, at least 92% identity, at least 93% identity, atleast 94% identity, at least 95% identity, such as at least 96%identity, 97% identity, 98% identity, or 99% identity to any of theextracellular domain (ECD) set forth in SEQ ID NOS: 387-424, 427-433,435-470 and contains the amino acid modification(s), e.g.substitution(s) not present in the reference (e.g., unmodified) orwild-type ICOSL. In some embodiments, the variant ICOSL polypeptidecomprises a specific binding fragment of any of the extracellular domain(ECD) sequences set forth in SEQ ID NOS: 387-424, 427-433, 435-470 andcontains the amino acid modification(s), e.g. substitution (s) notpresent in the reference (e.g., unmodified) or wild-type ICOSL. In someembodiments, the variant ICOSL polypeptide comprises any of the IgVsequences set forth in SEQ ID NOS: 425-426. In some embodiments, thevariant ICOSL polypeptide comprises a polypeptide sequence that exhibitsat least 90% identity, at least 91% identity, at least 92% identity, atleast 93% identity, at least 94% identity, at least 95% identity, suchas at least 96% identity, 97% identity, 98% identity, or 99% identity toany of the IgV sequences set forth in SEQ ID NO: 425-426 and containsthe amino acid modification(s), e.g. substitution(s) not present in thereference (e.g., unmodified) or wild-type ICOSL. In some embodiments,the variant ICOSL polypeptide comprises a specific binding fragment ofany of the IgV sequences set forth in SEQ ID NO: 425-426 and containsthe amino acid substitution(s) not present in the reference (e.g.,unmodified) or wild-type ICOSL.

III. FORMAT OF VARIANT POLYPEPTIDES

The immunomodulatory polypeptide comprising a variant ICOSL providedherein can be formatted in a variety of ways, including as a solubleprotein, a membrane bound protein, secreted protein, conjugate or fusionor for expression by an engineered cell or infectious agent as describedelsewhere herein. In some aspects, both immunomodulatory polypeptidescomprising one or more vIgD of ICOSL or immunomodulatory polypeptidescomprising multiple IgSF domains can be formatted in a variety of ways.

In some embodiments, the particular format can be chosen for the desiredtherapeutic application. In some cases, an immunomodulatory polypeptidecomprising a variant ICOSL polypeptide is provided in a format toantagonize or block activity of its cognate binding partner, e.g. CD28.In some embodiments, antagonism of CD28 may be useful to treatinflammation or autoimmunity. In some cases, an immunomodulatorypolypeptide comprising a variant ICOSL polypeptide is provided in aformat to agonize or stimulate activity of its cognate binding partner,e.g. CD28. In some embodiments, agonism of CD28 may be useful fortreating oncology indications. A skilled artisan can readily determinethe activity of a particular format, such as for antagonizing oragonizing one or more specific cognate binding partner. Exemplarymethods for assessing such activities are provided herein, including inthe examples.

In some embodiments, a soluble immunomodulatory polypeptide, such as avariant ICOSL containing a vIgD, can be encapsulated within a liposomewhich itself can be conjugated to any one of or any combination of theprovided conjugates (e.g., a targeting moiety). In some embodiments, thesoluble or membrane bound immunomodulatory polypeptides of the inventionare deglycosylated. In more specific embodiments, the variant ICOSLsequence is deglycosylated. In even more specific embodiments, the IgVand/or IgC (e.g. IgC2) domain or domains of the variant ICOSL isdeglycosylated.

Non-limiting examples of provided formats are described in FIGS. 13A-13Cand further described below.

A. Soluble Polypeptides

In some aspects, provided are immunomodulatory polypeptides comprising avIgD of ICOSL. In some embodiments, the immunomodulatory proteincontaining a variant ICOSL polypeptide is a soluble protein. Those ofskill will appreciate that cell surface proteins typically have anintracellular, transmembrane, and extracellular domain (ECD) and that asoluble form of such proteins can be made using the extracellular domainor an immunologically active subsequence thereof. Thus, in someembodiments, the immunomodulatory protein containing a variant ICOSLpolypeptide lacks a transmembrane domain or a portion of thetransmembrane domain. In some embodiments, the immunomodulatory proteincontaining a variant ICOSL lacks the intracellular (cytoplasmic) domainor a portion of the intracellular domain. In some embodiments, theimmunomodulatory protein containing the variant ICOSL polypeptide onlycontains the vIgD portion containing the ECD domain or a portion thereofcontaining an IgV domain and/or IgC (e.g. IgC2) domain or domains orspecific binding fragments thereof containing the amino acidmodification(s).

In some embodiments, an immunomodulatory polypeptide comprising avariant ICOSL can include one or more variant ICOSL polypeptides. Insome aspects, one or more additional IgSF domain, such as one or moreadditional vIgD, may be linked to a vIgD of ICOSL as provided herein. Insome aspects, both immunomodulatory polypeptides comprising one or morevIgD of ICOSL or immunomodulatory polypeptides comprising multiple IgSFdomains can be formatted in a variety of ways, such as described insubsection C of Section III.

In some embodiments, an immunomodulatory polypeptide comprising avariant ICOSL can include one or more variant ICOSL polypeptides of theinvention. In some embodiments a polypeptide of the invention willcomprise exactly 1, 2, 3, 4, 5 variant ICOSL sequences. In someembodiments, at least two of the variant ICOSL sequences are identicalvariant ICOSL sequences.

In some embodiments, the provided immunomodulatory polypeptide comprisestwo or more vIgD sequences of ICOSL. Multiple variant ICOSL polypeptideswithin the polypeptide chain can be identical (i.e., the same species)to each other or be non-identical (i.e., different species) variantICOSL sequences. In addition to single polypeptide chain embodiments, insome embodiments two, three, four, or more of the polypeptides of theinvention can be covalently or non-covalently attached to each other.Thus, monomeric, dimeric, and higher order (e.g., 3, 4, 5, or more)multimeric proteins are provided herein. For example, in someembodiments exactly two polypeptides of the invention can be covalentlyor non-covalently attached to each other to form a dimer. In someembodiments, attachment is made via interchain cysteine disulfide bonds.Compositions comprising two or more polypeptides of the invention can beof an identical species or substantially identical species ofpolypeptide (e.g., a homodimer) or of non-identical species ofpolypeptides (e.g., a heterodimer). A composition having a plurality oflinked polypeptides of the invention can, as noted above, have one ormore identical or non-identical variant ICOSL polypeptides of theinvention in each polypeptide chain.

In some aspects, one or more additional IgSF domain, such as one or moreadditional vIgD, may be linked to a vIgD of ICOSL as provided herein(hereinafter called a “stack” or “stacked” immunomodulatory protein). Insome embodiments, the modular format of the provided immunomodulatoryproteins provides flexibility for engineering or generatingimmunomodulatory proteins for modulating activity of multiplecounterstrucutres (multiple cognate binding partners). In someembodiments, such “stack” molecules can be provided in a soluble formator, in some cases, may be provided as membrane bound or secretedproteins.

In some embodiments, the immunomodulatory proteins can contain any ofthe variant ICOSL polypeptides provided herein linked, directly orindirectly, to one or more other immunoglobulin superfamily (IgSF)domain (“stacked” immunomodulatory protein construct and also called a“Type II” immunomodulatory protein). In some aspects, this can createunique multi-domain immunomodulatory proteins that bind two or more,such as three or more, cognate binding partners, thereby providing amulti-targeting modulation of the immune synapse.

In some embodiments, an immunomodulatory protein comprises a combination(a “non-wild-type combination”) and/or arrangement (a “non-wild typearrangement” or “non-wild-type permutation”) of a variant ICOSL domainwith one or more other affinity modified and/or non-affinity modifiedIgSF domain sequences of another IgSF family member (e.g. a mammalianIgSF family member) that are not found in wild-type IgSF family members.In some embodiments, the immunomodulatory protein contains 2, 3, 4, 5 or6 immunoglobulin superfamily (IgSF) domains, where at least one of theIgSF domain is a variant ICOSL IgSF domain (vIgD of ICOSL) according tothe provided description.

In some embodiments, the sequences of the additional IgSF domains can bea modified IgSF domain that contains one or more amino acidmodifications, e.g. substitutions, compared to a reference (e.g.,unmodified) or wild-type IgSF domain. In some embodiments, the IgSFdomain can be non-affinity modified (e.g., wild-type) or have beenaffinity modified. In some embodiments, the reference (e.g., unmodified)or wild-type IgSF domain can be from mouse, rat, cynomolgus monkey, orhuman origin, or combinations thereof. In some embodiments, theadditional IgSF domains can be an IgSF domain of an IgSF family memberset forth in Table 2. In some embodiments, the additional IgSF domaincan be an affinity-modified IgSF domain containing one or more aminoacid modifications, e.g. substitutions, compared to an IgSF domaincontained in an IgSF family member set forth in Table 2.

In some embodiments, the additional IgSF domain is an affinity ornon-affinity modified IgSF domain contained in an IgSF family member ofa family selected from Signal-Regulatory Protein (SIRP) Family,Triggering Receptor Expressed On Myeloid Cells Like (TREML) Family,Carcinoembryonic Antigen-related Cell Adhesion Molecule (CEACAM) Family,Sialic Acid Binding Ig-Like Lectin (SIGLEC) Family, Butyrophilin Family,B7 family, CD28 family, V-set and Immunoglobulin Domain Containing(VSIG) family, V-set transmembrane Domain (VS™) family, MajorHistocompatibility Complex (MHC) family, Signaling lymphocyticactivation molecule (SLAM) family, Leukocyte immunoglobulin-likereceptor (LIR), Nectin (Nec) family, Nectin-like (NECL) family,Poliovirus receptor related (PVR) family, Natural cytotoxicitytriggering receptor (NCR) family, T cell immunoglobulin and mucin (TIM)family or Killer-cell immunoglobulin-like receptors (KIR) family. Insome embodiments, the additional IgSF domains are independently derivedfrom an IgSF protein selected from the group consisting of CD80(B7-1),CD86(B7-2), CD274 (PD-L1, B7-H1), PDCD1LG2(PD-L2, CD273), ICOSLG (B7RP1,CD275, ICOSL, B7-H2), CD276(B7-H3), VTCN1(B7-H4), CD28, CTLA4,PDCD1(PD-1), ICOS, BTLA(CD272), CD4, CD8A(CD8-alpha), CD8B(CD8-beta),LAG3, HAVCR2(TIM-3), CEACAM1, TIGIT, PVR(CD155), PVRL2(CD112), CD226,CD2, CD160, CD200, CD200R1(CD200R), and NCR3 (NKp30).

The first column of Table 2 provides the name and, optionally, the nameof some possible synonyms for that particular IgSF member. The secondcolumn provides the protein identifier of the UniProtKB database, apublicly available database accessible via the internet at uniprot.orgor, in some cases, the GenBank Number. The Universal Protein Resource(UniProt) is a comprehensive resource for protein sequence andannotation data. The UniProt databases include the UniProt Knowledgebase(UniProtKB). UniProt is a collaboration between the EuropeanBioinformatics Institute (EMBL-EBI), the SIB Swiss Institute ofBioinformatics and the Protein Information Resource (PIR) and supportedmainly by a grant from the U.S. National Institutes of Health (NIH).GenBank is the NIH genetic sequence database, an annotated collection ofall publicly available DNA sequences (Nucleic Acids Research, 2013January; 41(D1):D36-42). The third column provides the region where theindicated IgSF domain is located. The region is specified as a rangewhere the domain is inclusive of the residues defining the range. Column3 also indicates the IgSF domain class for the specified IgSF region.Column 4 provides the region where the indicated additional domains arelocated (signal peptide, S; extracellular domain, E; transmembranedomain, T; cytoplasmic domain, C). It is understood that description ofdomains can vary depending on the methods used to identify or classifythe domain, and may be identified differently from different sources.The description of residues corresponding to a domain in Table 2 is forexemplification only and can be several amino acids (such as one, two,three or four) longer or shorter. Column 5 indicates for some of thelisted IgSF members, some of its cognate cell surface binding partners.

TABLE 2 IgSF members according to the present disclosure. NCBI ProteinAccession IgSF Member Amino Acid Sequence Number/ (SEQ ID NO) IgSFUniProtKB Cognate Cell Precursor Member Protein IgSF Region & OtherSurface Binding (mature (Synonyms) Identifier Domain Class DomainsPartners residues) Mature ECD CD80 NP_005182.1 35-135, S: 1-34, CD28,CTLA4, SEQ ID SEQ ID SEQ ID (B7-1) P33681 35-138 or E: 35-242, PD-L1 NO:1 NO: 253 NO: 28 35-141, T: 243-263, (35-288) 37-138 IgV, C: 264-288145-230 or 154-232 IgC CD86 P42081.2 33-131 IgV, S: 1-23, CD28, CTLA4SEQ ID SEQ ID SEQ ID (B7-2) 150-225 IgC2 E: 24-247, NO: 2 NO: 254 NO: 29T: 248-268, (24-329) C: 269-329 CD274 Q9NZQ7.1 24-130, S: 1-18, PD-1,B7-1 SEQ ID SEQ ID SEQ ID (PD-L1, 19-127, IgV, E: 19-238, NO: 3 NO: 255NO: 30 B7-H1) 133-225 IgC2 T: 239-259, (19-290) C: 260-290 PDCD1LG2Q9BQ51.2 21-118 IgV, S: 1-19, PD-1, RGMb SEQ ID SEQ ID SEQ ID (PD-L2,122-203 IgC2 E: 20-220, NO: 4 NO: 256 NO: 31 CD273) T: 221-241, (20-273)C: 242-273 ICOSLG O75144.2 19-129 IgV, S: 1-18, ICOS, CD28, SEQ ID SEQID SEQ ID (B7RP1, 141-227 IgC2 E: 19-256, CTLA4 NO: 5 NO: 257 NO: 32CD275, T: 257-277, (19-302) ICOSL, C: 278-302 B7-H2) CD276 Q5ZPR3.129-139 IgV, S: 1-28, SEQ ID SEQ ID SEQ ID (B7-H3) 145-238 IgC2, E:29-466, NO: 6 NO: 258 NO: 33 243-357 IgV2, T: 467-487, (29-534) 367-453,C: 488-534 363-456 IgC2 VTCN1 Q7Z7D3.1 35-146 IgV, S: 1-24, SEQ ID SEQID SEQ ID (B7-H4) 153-241 IgV E: 25-259, NO: 7 NO: 259 NO: 34 T:260-280, (25-282) C: 281-282 CD28 P10747.1 28-137 IgV S: 1-18, B7-1,B7-2, SEQ ID SEQ ID SEQ ID E: 19-152, B7RP1 NO: 8 NO: 260 NO: 35 T:153-179, (19-220) C: 180-220 CTLA4 AAL07473.1 39-152 IgV, S: 1-35, B7-1,B7-2, SEQ ID SEQ ID SEQ ID P16410.3 39-140 IgV E: 36-161, B7RP1 NO: 9NO: 261 NO: 36 T: 162-182, (36-223) C: 183-223 PDCD1 Q15116.3 35-145 IgVS: 1-20, PD-L1, PD-L2 SEQ ID SEQ ID SEQ ID (PD-1) E: 21-170, NO: 10 NO:262 NO: 37 T: 171-191, (21-288) C: 192-288 ICOS Q9Y6W8.1 30-132 IgV S:1-20, B7RP1 SEQ ID SEQ ID SEQ ID E: 21-140, NO: 11 NO: 263 NO: 38 T:141-161, (21-199) C: 162-199 BTLA Q7Z6A9.3 31-132 IgV S: 1-30, HVEM SEQID SEQ ID SEQ ID (CD272) E: 31-157, NO: 12 NO: 264 NO: 39 T: 158-178,(31-289) C: 179-289 CD4 P01730.1 26-125 IgV, S: 1-25, MHC class II SEQID SEQ ID SEQ ID 126-203 IgC2, E: 26-396, NO: 13 NO: 265 NO: 40 204-317IgC2, T: 397-418, (26-458) 317-389, C: 419-458 318-374 IgC2 CD8AP01732.1 22-135 IgV S: 1-21, MHC class I SEQ ID SEQ ID SEQ ID (CD8- E:22-182, NO: 14 NO: 266 NO: 41 alpha) T: 183-203, (22-235) C: 204-235CD8B P10966.1 22-132 IgV S: 1-21, MHC class I SEQ ID SEQ ID SEQ ID (CD8-E: 22-170, NO: 15 NO: 267 NO: 42 beta) T: 171-191, (22-210) C: 192-210LAG3 P18627.5 37-167 IgV, S: 1-28, MHC class II SEQ ID SEQ ID SEQ ID168-252 IgC2, E: 29-450, NO: 16 NO: 268 NO: 43 265-343 IgC2, T: 451-471,(29-525) 349-419 IgC2 C: 472-525 HAVCR2 Q8TDQ0.3 22-124 IgV S: 1-21,CEACAM-1, SEQ ID SEQ ID SEQ ID (TIM-3) E: 22-202, phosphatidylserine,NO: 17 NO: 269 NO: 44 T: 203-223, Galectin-9, (22-301) C: 224-301 HMGB1CEACAM1 P13688.2 35-142 IgV, S: 1-34, TIM-3 SEQ ID SEQ ID SEQ ID 145-232IgC2, E: 35-428, NO: 18 NO: 270 NO: 45 237-317 IgC2, T: 429-452,(35-526) 323-413 IgC2 C: 453-526 TIGIT Q495A1.1 22-124 IgV S: 1-21,CD155, CD112 SEQ ID SEQ ID SEQ ID E: 22-141, NO: 19 NO: 271 NO: 46 T:142-162, (22-244) C: 163-244 PVR P15151.2 24-139 IgV, S: 1-20, TIGIT,CD226, SEQ ID SEQ ID SEQ ID (CD155) 145-237 IgC2, E: 21-343, CD96, NO:20 NO: 272 NO: 47 244-328 IgC2 T: 344-367, poliovirus (21-417) C:368-417 PVRL2 Q92692.1 32-156 IgV, S: 1-31, TIGIT, CD226, SEQ ID SEQ IDSEQ ID (CD112) 162-256 IgC2, E: 32-360, CD112R NO: 21 NO: 273 NO: 48261-345 IgC2 T: 361-381, (32-538) C: 382-538 CD226 Q15762.2 19-126 IgC2,S: 1-18, CD155, CD112 SEQ ID SEQ ID SEQ ID 135-239 IgC2 E: 19-254, NO:22 NO: 274 NO: 49 T: 255-275, (19-336) C: 276-336 CD2 P06729.2 25-128IgV, S: 1-24, CD58 SEQ ID SEQ ID SEQ ID 129-209 IgC2 E: 25-209, NO: 23NO: 275 NO: 50 T: 210-235, (25-351) C: 236-351 CD160 O95971.1 27-122 IgVS: 1-26 HVEM, MHC SEQ ID SEQ ID SEQ ID E: 27-122 family of NO: 24 NO:276 NO: 51 proteins (27-159) CD200 P41217.4 31-141 IgV, S: 1-30, CD200RSEQ ID SEQ ID SEQ ID 142-232 IgC2 E: 31-232, NO: 25 NO: 277 NO: 52 T:233-259, (31-278) C: 260-278 CD200R1 Q8TD46.2 53-139 IgV, S: 1-28, CD200SEQ ID SEQ ID SEQ ID (CD200R) 140-228 IgC2 E: 29-243, NO: 26 NO: 278 NO:53 T: 244-264, (29-325) C: 265-325 NCR3 O14931.1 19-126 IgC- S: 1-18,B7-H6 SEQ ID SEQ ID SEQ ID (NKp30) like E: 19-135, NO: 27 NO: 279 NO: 54T: 136-156, (19-201) C:157-201 VSIG8 Q5VU13 22-141 IgV1 S: 1-21 VISTASEQ ID SEQ ID SEQ ID 146-257 E: 22-263 NO: 341 NO: 342 NO: 343 IgV2 T:264-284 (22-414) C: 285-414

In some embodiments, the provided immunomodulatory proteins, in additionto containing a variant ICOSL polypeptide, also contains at least 1, 2,3, 4, 5 or 6 additional immunoglobulin superfamily (IgSF) domains, suchas an IgD domain of an IgSF family member set forth in Table 2.

In some embodiments, the provided immunomodulatory protein contains atleast one additional IgSF domain (e.g. second IgSF domain). In someembodiments, the provided immunomodulatory protein contains at least twoadditional IgSF domains (e.g. second and third IgSF domain). In someembodiments, the provided immunomodulatory protein contains at leastthree additional IgSF domains (e.g. second, third and fourth). In someembodiments, the provided immunomodulatory protein contains at leastfour additional IgSF domains (e.g. second, third, fourth and fifth). Insome embodiments, the provided immunomodulatory protein contains atleast five additional IgSF domains (e.g. second, third, fourth, fifthand sixth). In some embodiments, the provided immunomodulatory proteincontains at least six additional IgSF domains (e.g. second, third,fourth, fifth, sixth and seventh). In some embodiments, each of the IgSFdomains in the immunomodulatory protein are different. In someembodiments, at least one of the additional IgSF domain is the same asat least one other IgSF domain in the immunomodulatory protein. In someembodiments, each of the IgSF domains is from or derived from adifferent IgSF family member. In some embodiments, at least two of theIgSF domains is from or derived from the same IgSF family member.

In some embodiments, the additional IgSF domain comprises an IgV domainor an IgC (e.g., IgC2) domain or domains, or a specific binding fragmentof the IgV domain or a specific binding fragment of the IgC (e.g., IgC2)domain or domains. In some embodiments, the additional IgSF domain is orcomprises a full-length IgV domain. In some embodiments, the additionalIgSF domain is or comprises a full-length IgC (e.g., IgC2) domain ordomains. In some embodiments, the additional IgSF domain is or comprisesa specific binding fragment of the IgV domain. In some embodiments, theadditional IgSF domain is or comprises a specific binding fragment ofthe IgC (e.g., IgC2) domain or domains. In some embodiments, theimmunomodulatory protein contains at least two additional IgSF domainsfrom a single (same) IgSF member. For example, in some aspects, theimmunomodulatory protein contains an ECD or portion thereof of an IgSFmember containing a full-length IgV domain and a full-length IgC (e.g.,IgC2) domain or domains or specific binding fragments thereof.

In some embodiments, the provided immunomodulatory proteins contain atleast one additional IgSF domain (e.g. a second or, in some cases, alsoa third IgSF domain) in which at least one additional, e.g., second orthird IgSF domain, is an IgSF domain set forth in a reference (e.g.,unmodified) or wild-type IgSF domain or a specific binding fragmentthereof contained in the sequence of amino acids set forth in any of SEQID NOS: 1-27 and 341. In some embodiments, the reference (e.g.,unmodified) or wild-type IgSF domain is an IgV domain or an IgC domain,such as an IgC1 or IgC2 domain.

In some embodiments, the provided immunomodulatory proteins, in additionto containing a variant ICOSL polypeptide, also contains at least oneadditional IgSF domain (e.g. a or, in some cases, also a thirdaffinity-modified IgSF domain and so on) in which at least oneadditional IgSF domain is a vIgD that contains one or more amino acidmodifications (e.g. substitution, deletion or mutation) compared to anIgSF domain in a reference (e.g., unmodified) or wild-type IgSF domain,such as an IgSF domain in an IgSF family member set forth in Table 2. Insome embodiments, the additional, e.g., second or thirdaffinity-modified IgSF domain comprises at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to a reference (e.g., unmodified) or wild-type IgSF domain or aspecific binding fragment thereof contained in the sequence of aminoacids set forth in any of SEQ ID NOS: 1-27 and 341. In some embodiments,the reference (e.g., unmodified) or wild-type IgSF domain is an IgVdomain or an IgC domain, such as an IgC1 or IgC2 domain. In someembodiments, the additional, e.g., second or third IgSF domain is anaffinity-modified IgV domain and/or IgC domain. In some embodiments, theone or more additional IgSF domain is an affinity-modified IgSF domainthat contains an IgV domain and/or an IgC (e.g., IgC2) domain ordomains, or a specific binding fragment of the IgV domain and/or aspecific binding fragment of the IgC (e.g., IgC2) domain or domains, inwhich the IgV and/or IgC domain contains the amino acid modification(s)(e.g., substitution(s)). In some embodiments, the one or more additionalaffinity-modified IgSF domain contains an IgV domain containing theamino acid modification(s) (e.g. substitution(s)). In some embodiments,the one or more additional affinity-modified IgSF domain include IgSFdomains present in the ECD or a portion of the ECD of the correspondingreference IgSF family member, such as a full-length IgV domain and afull-length IgC (e.g., IgC2) domain or domains, or specific bindingfragments thereof, in which one or both of the IgV and IgC contain theamino acid modification(s) (e.g. substitution(s)). In some embodiments,the particular domain or each of the particular domains (e.g.additional, e.g., second or third IgSF domain) of a variant IgSF domainpolypeptide can be several amino acids longer or shorter, such as 1-10,e.g., 1, 2, 3, 4, 5, 6 or 7 amino acids longer or shorter, than thesequence of amino acids set forth in the respective SEQ ID NO.

In some embodiments, the provided immunomodulatory protein contains atleast one additional, (e.g., second or, in some cases, also a third IgSFdomain and so on) or second IgSF domain that is a vIgD that contains oneor more amino acid substitutions compared to an IgSF domain (e.g., IgV)of a reference (e.g., unmodified) or wild-type IgSF domain other thanICOSL.

In some embodiments, the additional or second IgSF domain contains oneor more amino acid substitutions compared to an IgSF domain in areference (e.g., unmodified) or wild-type IgSF domain, such as an IgSFdomain in an IgSF family member set forth in Table 2. In someembodiments, the additional or second affinity-modified IgSF domaincomprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to a reference (e.g.,unmodified) or wild-type IgSF domain or a specific binding fragmentthereof contained in the sequence of amino acids set forth in any of SEQID NOS: 1-27. In some embodiments, the reference (e.g., unmodified) orwild-type IgSF domain is an IgV domain or an IgC domain, such as an IgC1or IgC2 domain. In some embodiments, the additional or second IgSFdomain is an affinity-modified IgV domain or IgC domain. Tables 3-5provide exemplary polypeptides containing one or more affinity-modifiedIgSF domains that can be used in stack constructs provided herein.

In some embodiments, the one or more additional IgSF domain (e.g. secondIgSF) domain is an IgSF domain (e.g. IgV) of another IgSF family memberthat binds or recognizes a tumor antigen. In such embodiments, the IgSFfamily member serves as a tumor-localizing moiety, thereby bringing thevIgD of ICOSL in close proximity to immune cells in the tumormicroenvironment. In some embodiments, the additional IgSF domain (e.g.second IgSF) domain is an IgSF domain of NKp30, which binds orrecognizes B7-H6 expressed on a tumor cell. In some embodiments, the atleast one additional (e.g. second) IgSF domain, e.g. NKp30, is a vIgDthat contains one or more amino acid modifications (e.g. substitutions,deletions or additions). In some embodiments, the one or more amino acidmodifications increase binding affinity and/or selectivity to B7-H6compared to reference IgSF domain, e.g. NKp30, such as by at least or atleast about 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold 40-fold or 50-fold.

TABLE 3 Exemplary variant CD80 polypeptides ECD IgV SEQ ID SEQ IDMutation(s) NO NO Wild-type 28 152 L70Q/A91G 55 153 L70Q/A91G/T130A 56L70Q/A91G/I118A/T120S/T130A 57 V4M/L70Q/A91G/T120S/T130A 58 154L70Q/A91G/T120S/T130A 59 V20L/L70Q/A91S/T120S/T130A 60 155S44P/L70Q/A91G/T130A 61 156 L70Q/A91G/E117G/T120S/T130A 62A91G/T120S/T130A 63 157 L70R/A91G/T120S/T130A 64 158L70Q/E81A/A91G/T120S/I127T/T130A 65 159 L70Q/Y87N/A91G/T130A 66 160T28S/L70Q/A91G/E95K/T120S/T130A 67 161 N63S/L70Q/A91G/T120S/T130A 68 162K36E/I67T/L70Q/A91G/T120S/T130A/ 69 163 N152T E52G/L70Q/A91G/T120S/T130A70 164 K37E/F59S/L70Q/A91G/T120S/T130A 71 165 A91G/S103P 72 K89E/T130A73 166 A91G 74 D60V/A91G/T120S/T130A 75 167 K54M/A91G/T120S 76 168M38T/L70Q/E77G/A91G/T120S/T130A/ 77 169 N152TR29H/E52G/L70R/E88G/A91G/T130A 78 170 Y31H/T41G/L70Q/A91G/T120S/T130A 79171 V68A/T110A 80 172 S66H/D90G/T110A/F116L 81 173 R29H/E52G/T120S/T130A82 174 A91G/L102S 83 I67T/L70Q/A91G/T120S 84 175L70Q/A91G/T110A/T120S/T130A 85 M38V/T41D/M43I/W50G/D76G/V83A/ 86 176K89E/T120S/T130A V22A/L70Q/S121P 87 177 A12V/S15F/Y31H/T41G/T130A/ 88178 P137L/152T I67F/L70R/E88G/A91G/T120S/ 89 179 T130AE24G/L25P/L70Q/T120S 90 180 A91G/F92L/F108L/T120S 91 181R29D/Y31L/Q33H/K36G/M38I/ 92 182 T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/ I118T/N149S R29D/Y31L/Q33H/K36G/M38I/ 93T41A/M43R/M47T/E81V/L85R/ K89N/A91T/F92P/K93V/R94L/ N144S/N149SR29D/Y31L/Q33H/K36G/M38I/ 94 183 T41A/M42T/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/ R94L/L148S/N149S E24G/R29D/Y31L/Q33H/K36G/ 95184 M38I/T41A/M43R/M47T/F59L/ E81V/L85R/K89N/A91T/F92P/K93V/R94L/H96R/N149S/C182S R29D/Y31L/Q33H/K36G/M38I/ 96T41A/M43R/M47T/E81V/L85R/ K89N/A91T/F92P/K93V/R94L/ N149SR29V/M43Q/E81R/L85I/K89R/ 97 185 D90L/A91E/F92N/K93Q/R94G T41I/A91G 98186 K89R/D90K/A91G/F92Y/K93R/ 99 187 N122S/N177SK89R/D90K/A91G/F92Y/K93R 100 K36G/K37Q/M38I/F59L/E81V/ 101 188L85R/K89N/A91T/F92P/K93V/ R94L/E99G/T130A/N149SE88D/K89R/D90K/A91G/F92Y/ 102 189, 543 K93R K36G/K37Q/M38I/L40M 103 190K36G 104 191 R29H/Y31H/T41G/Y87N/E88G/ 105 192 K89E/D90N/A91G/P109SA12T/H18L/M43V/F59L/E77K/ 106 193 P109S/I118T R29V/Y31F/K36G/M38L/M43Q/107 194 E81R/V83I/L85I/K89R/D90L/ A91E/F92N/K93Q/R94GV68M/L70P/L72P/K86E 108 195 L70Q/A91G/N144D 508 L70Q/A91G/I118A/T120S/509 T130A/K169E V4M/L70Q/A91G/I118V/T120S/ 510 T130A/K169EL70Q/A91G/I118V/T120S/T130A/ 511 K169E L70Q/A91G/I118V/T120S/T130A 512V20L/L70Q/A91S/I118V/T120S/ 513 T130A L70Q/A91G/E117G/I118V/T120S/ 514T130A A91G/I118V/T120S/T130A 515 L70R/A91G/I118V/T120S/T130A/ 516 T199SL70Q/E81A/A91G/I118V/T120S/ 517 I127T/T130A T28S/L70Q/A91G/E95K/I118V/518 T120S/I126V/T130A/K169E N63S/L70Q/A91G/S114T/I118V/ 519 T120S/T130AK36E/I67T/L70Q/A91G/I118V/ 520 T120S/T130A/N152TE52G/L70Q/A91G/D107N/I118V/ 521 T120S/T130A/K169EK37E/F59S/L70Q/A91G/I118V/ 522 T120S/T130A/K185E D60V/A91G/I118V/T120S/523 T130AK169E K54M/L70Q/A91G/Y164H/T120S 524 M38T/L70Q/E77G/A91G/I118V/525 T120S/T130A/N152T Y31H/T41G/M43L/L70Q/A91G/ 526I118V/T120S/I126V/T130A LS656H/D90G/T110A/F116L 527R29H/E52G/D90N/I118V/T120S/ 528 T130A R29H/E52G/D90N/I118V/T120S/ 529T130A I67T/L70Q/A91G/I118V/T120S 530 L70Q/A91G/T110A/I118V/T120S/ 531T130A M38V/T41D/M43I/W50G/D76G/ 532 V83A/K89E/I118V/T120S/ I126V/T130AA12V/S15F/Y31H/M38L/T41G/ 533 M43L/D90N/T130A/P137L/ N149D/N152TI67F/L70R/E88G/A91G/I118V/ 534 T120S/T130A E24G/L25P/L70Q/A91G/I118V/535 T120S/N152T A91G/F92L/F108L/I118V/ 536 T120SE88D/K89R/D90K/A91G/F92Y/ 537 K93R/N122S/N177S K36G/K37Q/M38I/L40M/F59L/539 E81V/L85R/K89N/A91T/F92P/ K93V/R94L/E99G/T130A/N149S K36G/L40M 540542, 544

TABLE 4 Exemplary variant NKp30 polypeptides IgC-like IgV-like ECDdomain domain SEQ ID SEQ ID SEQ ID Mutation(s) NO NO NO Wild-type 54 214929 L30V/A60V/S64P/S86G 143 215 504 L30V 144 216 930 A60V 145 217 931S64P 146 218 932 S86G 147 219 933

TABLE 5 Exemplary variant CD86 polypeptides ECD IgV SEQ ID SEQ IDMutation(s) NO NO Wild-type 29 220 Q35H/H90L/Q102H 148 221 Q35H 149 222H90L 150 223 Q102H 151 224

The number of such non-affinity modified or affinity modified IgSFdomains present in a “stacked” immunomodulatory protein construct(whether non-wild type combinations or non-wild type arrangements) is atleast 2, 3, 4, or 5 and in some embodiments exactly 2, 3, 4, or 5 IgSFdomains (whereby determination of the number of affinity modified IgSFdomains disregards any non-specific binding fractional sequences thereofand/or substantially immunologically inactive fractional sequencesthereof).

In some embodiments of a stacked immunomodulatory protein providedherein, the number of IgSF domains is at least 2 wherein the number ofaffinity modified and the number of non-affinity modified IgSF domainsis each independently at least: 0, 1, 2, 3, 4, 5, or 6. Thus, the numberof affinity modified IgSF domains and the number of non-affinitymodified IgSF domains, respectively, (affinity modified IgSF domain:non-affinity modified IgSF domain), can be exactly or at least: 2:0(affinity modified:wild-type), 0:2, 2:1, 1:2, 2:2, 2:3, 3:2, 2:4, 4:2,1:1, 1:3, 3:1, 1:4, 4:1, 1:5, or 5:1.

In some embodiments of a stacked immunomodulatory protein, at least twoof the non-affinity modified and/or affinity modified IgSF domains areidentical IgSF domains.

In some embodiments, a stacked immunomodulatory protein provided hereincomprises at least two affinity modified and/or non-affinity modifiedIgSF domains from a single IgSF member but in a non-wild-typearrangement (alternatively, “permutation”). One illustrative example ofa non-wild type arrangement or permutation is an immunomodulatoryprotein comprising a non-wild-type order of affinity modified and/ornon-affinity modified IgSF domain sequences relative to those found inthe wild-type ICOSL whose IgSF domain sequences served as the source ofthe variant IgSF domains as provided herein. Thus, in one example, theimmunomodulatory protein can comprise an IgV proximal and an IgC distalto the transmembrane domain albeit in a non-affinity modified and/oraffinity modified form. The presence, in an immunomodulatory proteinprovided herein, of both non-wild-type combinations and non-wild-typearrangements of non-affinity modified and/or affinity modified IgSFdomains is also within the scope of the provided subject matter.

In some embodiments of a stacked immunomodulatory protein, thenon-affinity modified and/or affinity modified IgSF domains arenon-identical (i.e., different) IgSF domains. Non-identical affinitymodified IgSF domains specifically bind, under specific bindingconditions, different cognate binding partners and are “non-identical”irrespective of whether or not the reference (e.g., unmodified) orwild-type IgSF domains from which they are engineered was the same.Thus, for example, a non-wild-type combination of at least twonon-identical IgSF domains in an immunomodulatory protein can compriseat least one IgSF domain sequence whose origin is from and unique to oneICOSL, and at least one of a second IgSF domain sequence whose origin isfrom and unique to another IgSF family member that is not ICOSL, whereinthe IgSF domains of the immunomodulatory protein are in non-affinitymodified and/or affinity modified form. However, in alternativeembodiments, the two non-identical IgSF domains originate from the sameIgSF domain sequence but at least one is affinity modified such thatthey specifically bind to different cognate binding partners.

A plurality of non-affinity modified and/or affinity modified IgSFdomains in a stacked immunomodulatory protein polypeptide chain need notbe covalently linked directly to one another. In some embodiments, anintervening span of one or more amino acid residues indirectlycovalently bonds the non-affinity modified and/or affinity modified IgSFdomains to each other. The linkage can be via the N-terminal toC-terminal residues.

In some embodiments, the linkage can be made via side chains of aminoacid residues that are not located at the N-terminus or C-terminus ofthe non-affinity modified and/or affinity modified IgSF domain. Thus,linkages can be made via terminal or internal amino acid residues orcombinations thereof.

In some embodiments, the two or more IgSF domain, including a vIgD ofICOSL and one or more additional IgSF domain (e.g. second or thirdvariant IgSF domain) from another IgSF family member, are covalently ornon-covalently linked. In some embodiments, the two or more IgSF domainsare linked directly or indirectly, such as via a linker. In someembodiments, an intervening span of one or more amino acid residuesindirectly covalently bonds IgSF domains to each other. The linkage canbe via the N-terminal to C-terminal residues. In some embodiments, thelinkage can be made via side chains of amino acid residues that are notlocated at the N-terminus or C-terminus of the IgSF domain(s). Thus,linkages can be made via terminal or internal amino acid residues orcombinations thereof.

In some embodiments, the immunomodulatory protein contains at least twoIgSF domains, each linked directly or indirectly via a linker. In someembodiments, the immunomodulatory protein contains at least threeimmunomodulatory proteins, each linked directly or indirectly via alinker. Various configurations are shown in FIGS. 16A and 16B.

In some embodiments, one or more “peptide linkers” link the vIgD ofICOSL and one or more additional IgSF domain (e.g. second or thirdvariant IgSF domain). In some embodiments, a peptide linker can be asingle amino acid residue or greater in length. In some embodiments, thepeptide linker has at least one amino acid residue but is no more than20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1amino acid residues in length. In some embodiments, the linker is aflexible linker. In some embodiments, the linker is (in one-letter aminoacid code): GGGGS (“4GS”; SEQ ID NO: 636) or multimers of the 4GSlinker, such as repeats of 2, 3, 4, or 5 4GS linkers. In someembodiments, the peptide linker is (GGGGS)₂ or (GGGGS)₃ as set forth inSEQ ID NOs: 229 and 228, respectively. In some embodiments, the linkeralso can include a series of alanine residues alone or in addition toanother peptide linker (such as a 4GS linker or multimer thereof). Insome embodiments, the number of alanine residues in each series is: 2,3, 4, 5, or 6 alanines. In some embodiments, the linker is a rigidlinker. For example, the linker is an α-helical linker. In someembodiments, the linker is (in one-letter amino acid code): EAAAK ormultimers of the EAAAK linker, such as repeats of 2, 3, 4, or 5 EAAAKlinkers, such as set forth in SEQ ID NO: 629 (1×EAAAK), SEQ ID NO: 630(3×EAAAK) or SEQ ID NO: 631 (5×EAAAK). In some embodiments, the linkercan further include amino acids introduced by cloning and/or from arestriction site, for example the linker can include the amino acids GS(in one-letter amino acid code) as introduced by use of the restrictionsite BAMHI. In some embodiments, the linker (in one-letter amino acidcode) is GSGGGGS (SEQ ID NO: 635). In some examples, the linker is a2×GGGGS followed by three alanines (GGGGSGGGGSAAA; SEQ ID NO: 230).

In some embodiments, the non-affinity modified and/or affinity modifiedIgSF domains are linked by “wild-type peptide linkers” inserted at theN-terminus and/or C-terminus of a second non-affinity modified and/oraffinity modified IgSF domains. In some embodiments, there is present aleading peptide linker inserted at the N-terminus of the first IgSFdomain and/or a first trailing sequence inserted at the C-terminus ofthe first non-affinity modified and/or affinity modified IgSF domain. Insome embodiments, there is present a second leading peptide linkerinserted at the N-terminus of the second IgSF domain and/or a secondtrailing sequence inserted at the C-terminus of the second non-affinitymodified and/or affinity modified IgSF domain. When the first and secondnon-affinity modified and/or affinity modified IgSF domains are derivedfrom the same parental protein and are connected in the sameorientation, wild-type peptide linkers between the first and secondnon-affinity modified and/or affinity modified IgSF domains are notduplicated. For example, when the first trailing wild-type peptidelinker and the second leading wild-type peptide linker are the same, theType II immunomodulatory protein does not comprise either the firsttrailing wild-type peptide linker or the second leading wild-typepeptide linker.

In some embodiments, the Type II immunomodulatory protein comprises afirst leading wild-type peptide linker inserted at the N-terminus of thefirst non-affinity modified and/or affinity modified IgSF domain,wherein the first leading wild-type peptide linker comprises at least 5(such as at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ormore) consecutive amino acids from the intervening sequence in thewild-type protein from which the first non-affinity modified and/oraffinity modified IgSF domain is derived between the parental IgSFdomain and the immediately preceding domain (such as a signal peptide oran IgSF domain). In some embodiments, the first leading wild-typepeptide linker comprises the entire intervening sequence in thewild-type protein from which the first non-affinity modified and/oraffinity modified IgSF domain is derived between the parental IgSFdomain and the immediately preceding domain (such as a signal peptide oran IgSF domain).

In some embodiments, the Type II immunomodulatory protein furthercomprises a first trailing wild-type peptide linker inserted at theC-terminus of the first non-affinity modified and/or affinity modifiedIgSF domain, wherein the first trailing wild-type peptide linkercomprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more) consecutive amino acids from the interveningsequence in the wild-type protein from which the first non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately following domain (such as anIgSF domain or a transmembrane domain). In some embodiments, the firsttrailing wild-type peptide linker comprises the entire interveningsequence in the wild-type protein from which the first non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately following domain (such as anIgSF domain or a transmembrane domain).

In some embodiments, the Type II immunomodulatory protein furthercomprises a second leading wild-type peptide linker inserted at theN-terminus of the second non-affinity modified and/or affinity modifiedIgSF domain, wherein the second leading wild-type peptide linkercomprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more) consecutive amino acids from the interveningsequence in the wild-type protein from which the second non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately preceding domain (such as asignal peptide or an IgSF domain). In some embodiments, the secondleading wild-type peptide linker comprises the entire interveningsequence in the wild-type protein from which the second non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately preceding domain (such as asignal peptide or an IgSF domain).

In some embodiments, the Type II immunomodulatory protein furthercomprises a second trailing wild-type peptide linker inserted at theC-terminus of the second non-affinity modified and/or affinity modifiedIgSF domain, wherein the second trailing wild-type peptide linkercomprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more) consecutive amino acids from the interveningsequence in the wild-type protein from which the second non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately following domain (such as anIgSF domain or a transmembrane domain). In some embodiments, the secondtrailing wild-type peptide linker comprises the entire interveningsequence in the wild-type protein from which the second non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately following domain (such as anIgSF domain or a transmembrane domain).

Exemplary of a leading sequence and trailing sequence for a Type IIprotein containing a CD80 IgSF domain is set forth in SEQ ID NO:231 andSEQ ID NO:232. Exemplary of a leading sequence and trailing sequence fora Type II protein containing an ICOSL IgSF domain is set forth in SEQ IDNO: 233 and 234. Exemplary of a leading sequence and a trailing sequencefor a Type II protein containing a CD86 IgSF domain is set forth in anyof SEQ ID NOS: 236-238. Exemplary of a wild-type linker sequence for aType II protein containing an NKp30 IgSF domain is set forth in SEQ IDNO: 235.

1. Monovalent

Provided herein are immunomodulatory proteins containing a variant ICOSLpolypeptide that is monovalent. In some embodiments, the variant ICOSLpolypeptide of the monovalent immunomodulatory protein is linked,directly or indirectly, to a further moiety. In some embodiments, thefurther moiety is a protein, peptide, small molecule or nucleic acid. Insome embodiments, the monovalent immunomodulatory protein is a fusionprotein.

In some embodiments, the moiety is a half-life extending molecule.Exemplary of such half-life extending molecules include, but are notlimited to, albumin, an albumin-binding polypeptide, Pro/Ala/Ser (PAS),a C-terminal peptide (CTP) of the beta subunit of human chorionicgonadotropin, polyethylene glycol (PEG), long unstructured hydrophilicsequences of amino acids (XTEN), hydroxyethyl starch (HES), analbumin-binding small molecule, or a combination thereof.

In some embodiments, the immunomodulatory polypeptide comprising avariant ICOSL can include conformationally disordered polypeptidesequences composed of the amino acids Pro, Ala, and Ser (See e.g.,WO2008/155134; SEQ ID NO: 904). In some cases, the amino acid repeat isat least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues,wherein each repeat comprises (an) Ala, Ser, and Pro residue(s). Thus,provided herein is an immunomodulatory protein is a PASylated proteinwherein the variant ICOSL polypeptide is linked, directly or indirectlyvia a linker, to Pro/Ala/Ser (PAS). In some embodiments, one or moreadditional linker structures may be used.

In some embodiments, the moiety facilitates detection or purification ofthe variant ICOSL polypeptide. In some cases, the immunomodulatorypolypeptide comprises a tag or fusion domain, e.g. affinity orpurification tag, linked, directly or indirectly, to the N- and/orc-terminus of the ICOSL polypeptide. Various suitable polypeptide tagsand/or fusion domains are known, and include but are not limited to, apoly-histidine (His) tag, a FLAG-tag (SEQ ID NO: 865), a Myc-tag, andfluorescent protein-tags (e.g., EGFP, set forth in SEQ ID NOs: 858, 859,or 896). In some cases, the immunomodulatory polypeptide comprising avariant ICOSL comprises at least six histidine residues (set forth inSEQ ID NO: 864). In some cases, the immunomodulatory polypeptidecomprising a variant ICOSL further comprises various combinations ofmoieties. For example, the immunomodulatory polypeptide comprising avariant ICOSL further comprises one or more polyhistidine-tag and FLAGtag.

In some embodiments, the ICOSL polypeptide is linked to a modifiedimmunoglobulin heavy chain constant region (Fc) that remains inmonovalent form such as set forth in SEQ ID NO: 472.

2. Bivalent

In some embodiments, the immunomodulatory protein containing a variantICOSL is multivalent, such as bivalent. In aspects, the immunomodulatoryprotein is linked, directly or indirectly via a linker, to amultimerization domain. In some aspects, the mutlimerization domainincrease half-life of the molecule.

Interaction of two or more variant ICOSL polypeptides can be facilitatedby their linkage, either directly or indirectly, to any moiety or otherpolypeptide that are themselves able to interact to form a stablestructure. For example, separate encoded variant ICOSL polypeptidechains can be joined by multimerization, whereby multimerization of thepolypeptides is mediated by a multimerization domain. Typically, themultimerization domain provides for the formation of a stableprotein-protein interaction between a first variant ICOSL polypeptideand a second variant ICOSL polypeptide. Homo- or heteromultimericpolypeptides can be generated from co-expression of separate variantICOSL polypeptides. The first and second variant ICOSL polypeptides canbe the same or different.

In some embodiments, a multimerization domain includes any capable offorming a stable protein-protein interaction. The multimerizationdomains can interact via an immunoglobulin sequence (e.g. Fc domain; seee.g., International Patent Pub. Nos. WO 93/10151 and WO 2005/063816 US;U.S. Pub. No. 2006/0024298; U.S. Pat. No. 5,457,035); leucine zipper(e.g. from nuclear transforming proteins fos and jun or theproto-oncogene c-myc or from General Control of Nitrogen (GCN4)) (eee.g., Busch and Sassone-Corsi (1990) Trends Genetics, 6:36-40; Gentz etal., (1989) Science, 243:1695-1699); a hydrophobic region; a hydrophilicregion; or a free thiol which forms an intermolecular disulfide bondbetween the chimeric molecules of a homo- or heteromultimer. Inaddition, a multimerization domain can include an amino acid sequencecomprising a protuberance complementary to an amino acid sequencecomprising a hole, such as is described, for example, in U.S. Pat. No.5,731,168; International Patent Pub. Nos. WO 98/50431 and WO2005/063816; Ridgway et al. (1996) Protein Engineering, 9:617-621. Sucha multimerization region can be engineered such that steric interactionsnot only promote stable interaction, but further promote the formationof heterodimers over homodimers from a mixture of chimeric monomers.Generally, protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g., tyrosine or tryptophan). Compensatory cavities ofidentical or similar size to the protuberances are optionally created onthe interface of the second polypeptide by replacing large amino acidside chains with smaller ones (e.g., alanine or threonine). Exemplarymultimerization domains are described below.

The variant ICOSL polypeptide can be joined anywhere, but typically viaits N- or C-terminus, to the N- or C-terminus of a multimerizationdomain to form a chimeric polypeptide. The linkage can be direct orindirect via a linker. Also, the chimeric polypeptide can be a fusionprotein or can be formed by chemical linkage, such as through covalentor non-covalent interactions. For example, when preparing a chimericpolypeptide containing a multimerization domain, nucleic acid encodingall or part of a variant ICOSL polypeptide can be operably linked tonucleic acid encoding the multimerization domain sequence, directly orindirectly or optionally via a linker domain. In some cases, theconstruct encodes a chimeric protein where the C-terminus of the variantICOSL polypeptide is joined to the N-terminus of the multimerizationdomain. In some instances, a construct can encode a chimeric proteinwhere the N-terminus of the variant ICOSL polypeptide is joined to theN- or C-terminus of the multimerization domain.

A polypeptide multimer contains two chimeric proteins created bylinking, directly or indirectly, two of the same or different variantICOSL polypeptides directly or indirectly to a multimerization domain.In some examples, where the multimerization domain is a polypeptide, agene fusion encoding the variant ICOSL polypeptide and multimerizationdomain is inserted into an appropriate expression vector. The resultingchimeric or fusion protein can be expressed in host cells transformedwith the recombinant expression vector, and allowed to assemble intomultimers, where the multimerization domains interact to formmultivalent polypeptides. Chemical linkage of multimerization domains tovariant ICOSL polypeptides can be effected using heterobifunctionallinkers.

The resulting chimeric polypeptides, such as fusion proteins, andmultimers formed therefrom, can be purified by any suitable method suchas, for example, by affinity chromatography over Protein A or Protein Gcolumns. Where two nucleic acid molecules encoding differentpolypeptides are transformed into cells, formation of homo- andheterodimers will occur. Conditions for expression can be adjusted sothat heterodimer formation is favored over homodimer formation.

Immunoglobulin Domain

In some embodiments, the immunomodulatory protein comprises a variantICOSL polypeptide attached to an Fc region of an immunoglobulin(yielding an “immunomodulatory Fc fusion,” such as an “ICOSL-Fc variantfusion,” also termed a ICOSL vIgD-Fc fusion). In some embodiments, theICOSL-Fc variant fusion also comprises one or more additional IgSFdomain(s), such as one or more additional vIgD linked to a vIgD ofICOSL. In some embodiments, the attachment of the variant ICOSLpolypeptide or additional IgSF domain is at the N-terminus of the Fc. Insome embodiments, the attachment of the variant ICOSL or additional IgSFdomain polypeptide is at the C-terminus of the Fc. In some embodiments,two or more ICOSL or additional IgSF domain variant polypeptides (thesame or different) are independently attached at the N-terminus and atthe C-terminus.

In some embodiments, the Fc is murine or human Fc. In some embodiments,the Fc is a mammalian or human IgG1, IgG2, IgG3, or IgG4 Fc regions. Insome embodiments, the Fc is derived from IgG1, such as human IgG1. Insome embodiments, the Fc comprises the amino acid sequence set forth inSEQ ID NO: 226 or a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to SEQ ID NO: 226.

In some embodiments, the Fc region contains one more modifications toalter (e.g. reduce) one or more of its normal functions. In general, theFc region is responsible for effector functions, such ascomplement-dependent cytotoxicity (CDC) and antibody-dependent cellcytotoxicity (ADCC), in addition to the antigen-binding capacity, whichis the main function of immunoglobulins. Additionally, the FcRn sequencepresent in the Fc region plays the role of regulating the IgG level inserum by increasing the in vivo half-life by conjugation to an in vivoFcRn receptor. In some embodiments, such functions can be reduced oraltered in an Fc for use with the provided Fc fusion proteins.

In some embodiments, one or more amino acid modifications may beintroduced into the Fc region of an ICOSL-Fc variant fusion providedherein, thereby generating an Fc region variant. In some embodiments,the Fc region variant has decreased effector function. There are manyexamples of changes or mutations to Fc sequences that can alter effectorfunction. For example, WO 2000/42072, WO2006/019447, WO2012/125850,WO2015/107026, US2016/0017041 and Shields et al. J Biol. Chem. 9(2):6591-6604 (2001) describe exemplary Fc variants with improved ordiminished binding to FcRs. The contents of those publications arespecifically incorporated herein by reference.

In some embodiments, the provided variant ICOSL-Fc fusions comprise anFc region that exhibits reduced effector functions (also called inert Fcor effectorless Fc), which makes it a desirable candidate forapplications in which the half-life of the ICOSL-Fc variant fusion invivo is important yet certain effector functions (such as CDC and ADCC)are unnecessary or deleterious. In vitro and/or in vivo cytotoxicityassays can be conducted to confirm the reduction/depletion of CDC and/orADCC activities. For example, Fc receptor (FcR) binding assays can beconducted to ensure that the ICOSL-Fc variant fusion lacks FcγR binding(hence likely lacking ADCC activity), but retains FcRn binding ability.The primary cells for mediating ADCC, NK cells, express FcγRIII only,whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of invitro assays to assess ADCC activity of a molecule of interest isdescribed in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al.Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al.,Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337(see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).Alternatively, non-radioactive assay methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96™non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes etal. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays mayalso be carried out to confirm that the ICOSL-Fc variant fusion isunable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3cbinding ELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S.et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie,Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/halflife determinations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

ICOSL-Fc variant fusions with reduced effector function include thosewith substitution of one or more of Fc region residues 238, 265, 269,270, 297, 327 and 329 by EU numbering (U.S. Pat. No. 6,737,056). Such Fcmutants include Fc mutants with substitutions at two or more of aminoacid positions 265, 269, 270, 297 and 327 by EU numbering, including theso-called “DANA” Fc mutant with substitution of residues 265 and 297 toalanine (U.S. Pat. No. 7,332,581).

In some embodiments, the Fc region of ICOSL-Fc variant fusions has an Fcregion in which any one or more of amino acids at positions 234, 235,236, 237, 238, 239, 270, 297, 298, 325, and 329 (indicated by EUnumbering) are substituted with different amino acids compared to thenative Fc region. Such alterations of Fc region are not limited to theabove-described alterations, and include, for example, alterations suchas deglycosylated chains (N297A and N297Q), IgG1-N297G,IgG1-L234A/L235A, IgG1-L234A/L235E/G237A, IgG1-A325A/A330S/P331S,IgG1-C226S/C229S, IgG1-C226S/C229S/E233P/L234V/L235A,IgG1-E233P/L234V/L235A/G236del/S267K, IgG1-L234F/L235E/P331S,IgG1-S267E/L328F, IgG2-V234A/G237A, IgG2-H268Q/V309L/A330S/A331S,IgG4-L235A/G237A/E318A, and IgG4-L236E described in Current Opinion inBiotechnology (2009) 20 (6), 685-691; alterations such as G236R/L328R,L235G/G236R, N325A/L328R, and N325LL328R described in WO 2008/092117;amino acid insertions at positions 233, 234, 235, and 237 (indicated byEU numbering); and alterations at the sites described in WO 2000/042072.

Certain Fc variants with improved or diminished binding to FcRs aredescribed. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312,WO2006/019447 and Shields et al., J. Biol. Chem. 9(2): 6591-6604(2001).)

In some embodiments, there is provided a ICOSL-Fc variant fusioncomprising a variant Fc region comprising one or more amino acidsubstitutions which increase half-life and/or improve binding to theneonatal Fc receptor (FcRn). Antibodies with increased half-lives andimproved binding to FcRn are described in US2005/0014934A1 (Hinton etal.) or WO2015107026. Those antibodies comprise an Fc region with one ormore substitutions therein which improve binding of the Fc region toFcRn. Such Fc variants include those with substitutions at one or moreof Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312,317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 by EUnumbering, e.g., substitution of Fc region residue 434 (U.S. Pat. No.7,371,826).

In some embodiments, the Fc region of a ICOSL-Fc variant fusioncomprises one or more amino acid substitution E356D and M358L by EUnumbering. In some embodiments, the Fc region of a ICOSL-Fc variantfusion comprises one or more amino acid substitutions C220S, C226S,and/or C229S by EU numbering. In some embodiments, the Fc region of aICOSL variant fusion comprises one or more amino acid substitutionsR292C and V302C. See also Duncan & Winter, Nature 322:738-40 (1988);U.S. Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

In some embodiments, the wild-type IgG1 Fc can be the Fc set forth inSEQ ID NO: 226 having an allotype containing residues Glu (E) and Met(M) at positions 356 and 358 by EU numbering (e.g., f allotype). Inother embodiments, the wild-type IgG1 Fc contains amino acids of thehuman G1m1 allotype, such as residues containing Asp (D) and Leu (L) atpositions 356 and 358, e.g. as set forth in SEQ ID NO: 927. Thus, insome cases, an Fc provided herein can contain amino acid substitutionsE356D and M358L to reconstitute residues of allotype G1 ml (e.g., alphaallotype). In some aspects, a wild-type Fc is modified by one or moreamino acid substitutions to reduce effector activity or to render the Fcinert for Fc effector function. Exemplary effectorless or inertmutations include those described herein. Among effectorless mutationsthat can be included in an Fc of constructs provided herein are L234A,L235E and G237A by EU numbering. In some embodiments, a wild-type Fc isfurther modified by the removal of one or more cysteine residue, such asby replacement of the cysteine residues to a serine residue at position220 (C220S) by EU numbering. Exemplary inert Fc regions having reducedeffector function are set forth in SEQ ID NO: 633 or 477 and SEQ ID NO:474 or 637, which are based on allotypes set forth in SEQ ID NO: 226 orSEQ ID NO: 927, respectively. In some embodiments, an Fc region used ina construct provided herein can further lack a C-terminal lysineresidue.

In some embodiments, alterations are made in the Fc region that resultin diminished C1q binding and/or Complement Dependent Cytotoxicity(CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, andIdusogie et al., J. Immunol. 164: 4178-4184 (2000).

In some embodiments, there is provided a ICOSL-Fc variant fusioncomprising a variant Fc region comprising one or more amino acidmodifications, wherein the variant Fc region is derived from IgG1, suchas human IgG. In some embodiments, the variant Fc region is derived fromthe amino acid sequence set forth in SEQ ID NO: 226. In someembodiments, the Fc exhibits reduced effector function. In someembodiments, the Fc contains at least one amino acid substitution thatis N82G by numbering of SEQ ID NO: 226 (corresponding to N297G by EUnumbering). In some embodiments, the Fc further contains at least oneamino acid substitution that is R77C or V87C by numbering of SEQ ID NO:226 (corresponding to R292C or V302C by EU numbering). In someembodiments, the variant Fc region further comprises a C5S amino acidmodification by numbering of SEQ ID NO: 226 (corresponding to C220S byEU numbering). For example, in some embodiments, the variant Fc regioncomprises the following amino acid modifications: V297G and one or moreof the following amino acid modifications C220S, R292C or V302C by EUnumbering (corresponding to N82G and one or more of the following aminoacid modifications C5S, R77C or V87C with reference to SEQ ID NO:226),e.g. the Fc region comprises the sequence set forth in SEQ ID NO:476. Insome embodiments, the variant Fc region comprises one or more of theamino acid modifications C220S, L234A, L235E or G237A, e.g. the Fcregion comprises the sequence set forth in SEQ ID NO:477. In someembodiments, the variant Fc region comprises one or more of the aminoacid modifications C220S, E233P, L234V, L235A, G236del or S267K, e.g.the Fc region comprises the sequence set forth in SEQ ID NO:478. In someembodiments, the variant Fc comprises one or more of the amino acidmodifications C220S, L234A, L235E, G237A, E356D or M358L, e.g. the Fcregion comprises the sequence set forth in SEQ ID NO:474.

In some embodiments, the Fc region lacks the C-terminal lysinecorresponding to position 232 of the reference (e.g., unmodified) orwild-type Fc set forth in SEQ ID NO: 56 (corresponding to K447del by EUnumbering). In some embodiments, because the C-terminal lysine may bedifferentially removed during biosynthesis, removal of the C-terminallysine residue results in a more homogenous product when the protein isexpressed in cells. In some aspects, such an Fc region can additionallyinclude one or more additional modifications, e.g. amino acidsubstitutions, such as any as described. Exemplary of such an Fc regionis set forth in SEQ ID NO: 632, 633, 634, or 637.

In some embodiments, there is provided a ICOSL-Fc variant fusioncomprising a variant Fc region in which the variant Fc comprises thesequence of amino acids set forth in any of SEQ ID NOS:474, 476, 477,478, 507, 632, 633, 634, or 637 or a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 474,476, 477, 478, 507, 632, 633, 634, or 637. In some embodiments, the Fcexhibits reduced effector function.

In some embodiments, the Fc is derived from IgG2, such as human IgG2. Insome embodiments, the Fc comprises the amino acid sequence set forth inSEQ ID NO: 227 or a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to SEQ ID NO: 227.

In some embodiments, the Fc comprises the amino acid sequence set forthin SEQ ID NO: 505 or a sequence of amino acids that exhibits at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to SEQ ID NO: 505. In some embodiments,the IgG4 Fc is a stabilized Fc in which the CH3 domain of human IgG4 issubstituted with the CH3 domain of human IgG1 and which exhibitsinhibited aggregate formation, an antibody in which the CH3 and CH2domains of human IgG4 are substituted with the CH3 and CH2 domains ofhuman IgG1, respectively, or an antibody in which arginine at position409 indicated in the EU index proposed by Kabat et al. of human IgG4 issubstituted with lysine and which exhibits inhibited aggregate formation(see e.g. U.S. Pat. No. 8,911,726). In some embodiments, the Fc is anIgG4 containing the S228P mutation, which has been shown to preventrecombination between a therapeutic antibody and an endogenous IgG4 byFab-arm exchange (see e.g. Labrijin et al. (2009) Nat. Biotechnol.,27(8) 767-71.) In some embodiments, the Fc comprises the amino acidsequence set forth in SEQ ID NO: 506 or a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 506.

In some embodiments, the variant ICOSL polypeptide is directly linked tothe Fc sequence. In some embodiments, the variant ICOSL polypeptide isindirectly linked to the Fc sequence, such as via a linker. In someembodiments, one or more “peptide linkers” link the variant ICOSLpolypeptide and the Fc domain. In some embodiments, a peptide linker canbe a single amino acid residue or greater in length. In someembodiments, the peptide linker has at least one amino acid residue butis no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,5, 4, 3, 2, or 1 amino acid residues in length. In some embodiments, thelinker is three alanines (AAA). In some embodiments, the linker is (inone-letter amino acid code): GGGGS (“4GS”; SEQ ID NO:636) or multimersof the 4GS linker, such as repeats of 2, 3, 4, 5 or 6 4GS linkers, suchas set forth in SEQ ID NO: 229 (2×GGGGS) or SEQ ID NO: 228 (3×GGGGS). Insome embodiments, the linker is a rigid linker. For example, the linkeris an α-helical linker. In some embodiments, the linker is (inone-letter amino acid code): EAAAK or multimers of the EAAAK linker,such as repeats of 2, 3, 4, or 5 4GS linkers, such as set forth in SEQID NO: 629 (EAAAK) or SEQ ID NO: 630 (3×EAAAK) or SEQ ID NO: 631(5×EAAAK). In some embodiments, linkers start with one or more EAAAKunits and can be lengthened by addition of A, AA, AAA, AAAA, EAAAA andEAAAK sequences. In some embodiments, the linker can further includeamino acids introduced by cloning and/or from a restriction site, forexample the linker can include the amino acids GS (in one-letter aminoacid code) as introduced by use of the restriction site BAMHI. In someembodiments, the linker (in one-letter amino acid code) is GSGGGGS (SEQID NO: 635). In some examples, the linker is a 2×GGGGS followed by threealanines (GGGGSGGGGSAAA; SEQ ID NO: 230).

In some embodiments, the variant ICOSL-Fc fusion protein is a dimerformed by two variant ICOSL Fc polypeptides linked to an Fc domain. Insome specific embodiments, identical or substantially identical species(allowing for 3 or fewer N-terminus or C-terminus amino acid sequencedifferences) of ICOSL-Fc variant fusion polypeptides will be dimerizedto create a homodimer. In some embodiments, the dimer is a homodimer inwhich the two variant ICOSL Fc polypeptides are the same. Alternatively,different species of ICOSL-Fc variant fusion polypeptides can bedimerized to yield a heterodimer. Thus, in some embodiments, the dimeris a heterodimer in which the two variant ICOSL Fc polypeptides aredifferent.

In some embodiments, provided is a variant ICOSL-Fc fusion proteincontaining a variant ICOSL polypeptide that includes one or more aminoacid modifications in a reference ICOSL as described in Section II thatis linked, directly or indirectly, to an Fc region. In some cases, theC-terminus of the variant ICOSL polypeptide is joined to the N-terminusof the Fc region. In some embodiments, the variant ICOSL of an ICOSL-Fcfusion contains one or more amino acid modifications in the sequence ofamino acids the reference IgV domain set forth in SEQ ID NO:545. Inparticular cases, such an immunomodulatory protein contains variantICOSL polypeptide containing an IgV domain, such as an IgV domain setforth in any one of SEQ ID NOS: 197-199, 201-208, 210, 212, 240,326-340, 382-386, 425-426, 434, 546-599, 686-857, 906-907, 909-910, oran IgV domain that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% to any of SEQ ID NOS: 197-199,201-208, 210, 212, 240, 326-340, 382-386, 425-426, 434, 546-599,686-857, 906-907, 909-910 and contains the one or more amino acidmodifications of the respective SEQ ID NO. In some embodiments, thevariant ICOSL polypeptide has an IgSF domain (e.g. IgV domain) thatexhibits increased binding affinity to CD28 or ICOS, such as any of theamino acid modifications described herein. In some embodiments, thevariant ICOSL polypeptide has an IgSF domain (e.g. IgV domain)containing one or more amino acid modification, e.g. substitution in anreference ICOSL or specific binding fragment, corresponding toposition(s) 52, 57, or 100 with reference to numbering of SEQ ID NO: 32.In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution selected from N52A, N52C, N52D,N52G, N52H, N52L, N52K, N52M, N52P, N52Q, N52R, N52S, N52T, N52V, N52Y,N57A, N57E, N57F, N57H, N57K, N57L, N57M, N57P, N57Q, N57S, N57T, N57V,N57W, N57Y, Q100A, Q100D, Q100E, Q100G, Q100K, Q100L, Q100M, Q100N,Q100R, Q100P, Q100S, Q100T, or Q100V. Exemplary of such variantmolecules include any as described herein. In some embodiments, thevariant ICOSL polypeptide contains the amino acid modificationsN52H/N57Y/Q100R (e.g. is or includes an IgV domain set forth in SEQ IDNO: 565). In some embodiments, the variant ICOSL polypeptide containsthe amino acid modifications N52D (e.g. is or includes an IgV domain setforth in SEQ ID NO: 548). In some embodiments, the variant ICOSLpolypeptide contains the amino acid modifications N52H/Q100R (e.g. is orincludes an IgV domain set forth in SEQ ID NO: 567). In someembodiments, the variant ICOSL polypeptide contains the amino acidmodifications N52L/N57H/Q100R (e.g. is or includes an IgV domain setforth in SEQ ID NO: 761). In some embodiments, the variant ICOSLpolypeptide contains the amino acid modifications N52H/N57Y/Q100P (e.g.is or includes an IgV domain set forth in SEQ ID NO: 570).

In particular embodiments of such variant ICOSL-Fc fusion proteins, theFc polypeptide is a variant of a human IgG1 Fc region that exhibitsreduced effector functions, such as any as described. In someembodiments, the Fc region is a human IgG1 that contains the amino acidmodifications N297G, E233P/L234V/L235A/G236del/S267K orL234A/L235E/G237A, wherein the residue is numbered according to the EUindex of Kabat. In some embodiments, the variant IgG1 Fc region furthercontains the amino acid substitution C220S, wherein the residues arenumbered according to the EU index of Kabat. In some embodiments, the Fcregion contains K447del, wherein the residue is numbered according tothe EU index of Kabat. In some aspects, the Fc region contains thesequence of amino acid sequence set forth in any of SEQ ID NOS: 474,476, 477, 478, 633 or 637 or a sequence of amino acids that exhibits atleast 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to any of SEQ ID NOS: 474, 476, 477, 478, 633 or 637and contains the amino acid substitutions of the respective SEQ ID NO.The linkage between the variant ICOSL IgSF (e.g. IgV) polypeptide andthe Fc can be via a peptide linker, such as any as described. In someembodiments, the linker is GGGGS (“4GS”; SEQ ID NO: 636), SEQ ID NO: 229(2×GGGGS) or SEQ ID NO: 228 (3×GGGGS). In particular examples, theC-terminus of the variant ICOSL polypeptide is joined to the N-terminusof the Fc region, such that the order of components is variantICOSL-linker-Fc.

In some embodiments, there is provided a variant ICOSL-Fc fusionprotein, e.g. variant ICOSL-linker-Fc, containing a variant ICOSL IgVdomain set forth in SEQ ID NO:565, a linker set forth in SEQ ID NO:636and an Fc polypeptide set forth in SEQ ID NO: 637. In some embodiments,there is provided a variant ICOSL-Fc fusion protein, e.g. variantICOSL-linker-Fc, containing a variant ICOSL IgV domain set forth in SEQID NO:565, a linker set forth in SEQ ID NO:636 and an Fc polypeptide setforth in SEQ ID NO: 474. In some embodiments, there is provided avariant ICOSL-Fc fusion protein, e.g. variant ICOSL-linker-Fc,containing a variant ICOSL IgV domain set forth in SEQ ID NO:565, alinker set forth in SEQ ID NO:636 and an Fc polypeptide set forth in SEQID NO: 477. In some embodiments, there is provided a variant ICOSL-Fcfusion protein, e.g. variant ICOSL-linker-Fc, containing a variant ICOSLIgV domain set forth in SEQ ID NO:565, a linker set forth in SEQ IDNO:636 and an Fc polypeptide set forth in SEQ ID NO: 633.

In some embodiments, there is provided a variant ICOSL-Fc fusionprotein, e.g. variant ICOSL-linker-Fc, containing a variant ICOSL IgVdomain set forth in SEQ ID NO:565, a linker set forth in SEQ ID NO: 229and an Fc polypeptide set forth in SEQ ID NO: 637. In some embodiments,there is provided a variant ICOSL-Fc fusion protein, e.g. variantICOSL-linker-Fc, containing a variant ICOSL IgV domain set forth in SEQID NO:565, a linker set forth in SEQ ID NO:229 and an Fc polypeptide setforth in SEQ ID NO: 474. In some embodiments, there is provided avariant ICOSL-Fc fusion protein, e.g. variant ICOSL-linker-Fc,containing a variant ICOSL IgV domain set forth in SEQ ID NO:565, alinker set forth in SEQ ID NO: 229 and an Fc polypeptide set forth inSEQ ID NO: 477. In some embodiments, there is provided a variantICOSL-Fc fusion protein, e.g. variant ICOSL-linker-Fc, containing avariant ICOSL IgV domain set forth in SEQ ID NO:565, a linker set forthin SEQ ID NO:229 and an Fc polypeptide set forth in SEQ ID NO: 633.

In some embodiments, there is provided a variant ICOSL-Fc fusionprotein, e.g. variant ICOSL-linker-Fc, containing a variant ICOSL IgVdomain set forth in SEQ ID NO:565, a linker set forth in SEQ ID NO:228and an Fc polypeptide set forth in SEQ ID NO: 637. In some embodiments,there is provided a variant ICOSL-Fc fusion protein, e.g. variantICOSL-linker-Fc, containing a variant ICOSL IgV domain set forth in SEQID NO:565, a linker set forth in SEQ ID NO: 228 and an Fc polypeptideset forth in SEQ ID NO: 474. In some embodiments, there is provided avariant ICOSL-Fc fusion protein, e.g. variant ICOSL-linker-Fc,containing a variant ICOSL IgV domain set forth in SEQ ID NO:565, alinker set forth in SEQ ID NO:228 and an Fc polypeptide set forth in SEQID NO: 477. In some embodiments, there is provided a variant ICOSL-Fcfusion protein, e.g. variant ICOSL-linker-Fc, containing a variant ICOSLIgV domain set forth in SEQ ID NO: 565, a linker set forth in SEQ IDNO:228 and an Fc polypeptide set forth in SEQ ID NO: 633.

In some embodiments, there is provided a variant ICOSL IgSF Fc fusionprotein that has the sequence of amino acids set forth in SEQ ID NO:928, or a sequence of amino acids that exhibits at least 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to SEQ ID NO:928. In someembodiments, the variant ICOSL IgSF Fc fusion protein binds to CD28 andICOS, such as with increased binding affinity compared to reference(wild-type) ICOSL-Fc fusion protein. In some embodiments, the variantICOSL IgSF Fc fusion exhibits reduced Fc effector function compared tofusion with an Fc of a wild-type human IgG1.

In some embodiments, there is provided a multi-domain stackimmunomodulatory protein in which two or more IgSF domain, including avIgD of ICOSL and one or more additional IgSF domain (e.g. secondvariant IgSF domain) from another IgSF family member, are linked orattached to an Fc to form an Fc fusion, which, upon expression in a cellcan, in some aspects, produce a dimeric multi-domain stackimmunomodulatory protein. Thus, also provided are dimeric multi-domainimmunomodulatory proteins.

In some embodiments, the variant ICOSL polypeptide and one or moreadditional IgSF domain are independently linked, directly or indirectly,to the N- or C-terminus of an Fc region. In some embodiments, thevariant ICOSL polypeptide and at least one of the one or more additionalIgSF domain are linked, directly or indirectly, and one of the variantICOSL or and one of the one or more additional IgSF domain is alsolinked, directly or indirectly, to the N- or C-terminus of an Fc region.In some embodiments, the N- or C-terminus of the Fc region is linked tothe variant ICOSL polypeptide or the one or more additional IgSF domainand the other of the N- or C-terminus of the Fc region is linked to theother of the ICOSL variant or another of the one or more additional IgSFdomain. In some embodiments, linkage to the Fc is via a peptide linker,e.g. a peptide linker, such as described above. In some embodiments,linkage between the variant ICOSL and second IgSF domain is via apeptide linker, e.g. a peptide linker, such as described above. In someembodiments, linkage between the variant ICOSL and the one or moreadditional IgSF domain is via a peptide linker, e.g., a peptide linker,such as described above. In some embodiments, the vIgD of ICOSL, the oneor more additional IgSF domains, and the Fc domain can be linkedtogether in any of numerous configurations as depicted in FIGS. 16A and16B. In some embodiments, the ICOSL-Fc variant fusion can furthercontain a signal peptide, such as an exemplary signal peptide ascontained in the sequence of amino acids set forth in SEQ ID NO: 59 or225. Exemplary configurations are described in the Examples.

In some embodiments, the stacked immunomodulatory protein is a dimerformed by two immunomodulatory Fc fusion polypeptides. Also provided arenucleic acid molecules encoding any of the stacked immunomodulatoryproteins. In some embodiments, the dimeric multi-domain stackimmunomodulatory protein can be produced in cells by expression, or insome cases co-expression, of stack immunomodulatory Fc regionpolypeptides, such as described further below.

In some embodiments, the dimeric multi-domain stack immunomodulatoryprotein is divalent for each Fc subunit, monovalent for each subunit, ordivalent for one subunit and tetravalent for the other.

In some embodiments, the dimeric multi-domain stack immunomodulatoryprotein is a homodimeric multi-domain stack Fc protein. In someembodiments, the dimeric multi-domain stack immunomodulatory proteincomprises a first stack immunomodulatory Fc fusion polypeptide and asecond stack immunomodulatory Fc fusion polypeptide in which the firstand second polypeptide are the same. In some embodiments, the Fc portionof the polypeptide can be any Fc as described above.

In some embodiments, the multi-domain stack molecule contains a first Fcfusion polypeptide containing a variant ICOSL and a second fusion IgSFdomain and a second Fc polypeptide containing the variant ICOSL and thesecond IgSF domain. In some embodiments, the multi-domain stack moleculecontains a first Fc fusion polypeptide containing a variant ICOSL and asecond IgSF domain, and a third IgSF domain and a second Fc fusionpolypeptide containing the variant ICOSL, the second IgSF domain, andthe third IgSF domain. In some embodiments, the Fc portion of the firstand/or second fusion polypeptide can be any Fc as described above. Insome embodiments, the Fc portion or region of the first and secondfusion polypeptide is the same.

In some embodiments, there is provided an immunomodulatory protein thatis a ICOSL-NKp30 multi-domain stack containing any of the variant ICOSLpolypeptides and one or more IgF domains of NKp30, e.g. wild-type orunmodified NKp30, such as an IgV domain set forth in SEQ ID NO: 929 oran ECD or a binding portion thereof set forth in SEQ ID NO: 215 or abinding portion thereof. In some embodiments, there is provided animmunomodulatory protein containing any of the variant ICOSL polypeptideand one or more IgSF domains of a variant NKp30 containing one or moreamino acid modifications in the wild-type or unmodified sequence setforth in SEQ ID NO: 215 or 929. In some embodiments, the one or moreamino acid modifications (e.g. substitutions) include one or more ofL30V, A60V, S64P, S86G, such as 1, 2, 3 or 4 of such amino acidmodifications. In some aspects, a variant NKp30 of the multi-domainstack polypeptides is or includes a variant IgV domain, such as avariant IgV domain set forth in any of SEQ ID NOS: 504, 930, 931, 932 or933, or an IgV domain that has at least 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to any of SEQ ID NOS:504,930, 931, 932 or 933 and contains the one or more amino acidmodifications of the respective SEQ ID NO. In some aspects, a variantNKp30 of the multi-domain stack polypeptides is or includes a variantECD domain, such as a variant ECD set forth in any of SEQ ID NOS: 215,216, 217, 218 or 219, or an ECD domain that has at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to any of SEQID NOS: 215, 216, 217, 218 or 219 and contains the one or more aminoacid modifications of the respective SEQ ID NO.

In any of such embodiments of an ICOSL-NKp30 multi-domain stack, thevariant ICOSL polypeptide can include any described in Section IIcontaining a variant IgSF domain (e.g. IgV or ECD), such as includingany of the amino acid modifications set forth in a Table 1. In somecases, such an immunomodulatory protein contains a variant ICOSLpolypeptide containing an ECD domain, such as an ECD domain set forth inany one of SEQ ID NOS: 109-142, 239, 280-325, 364-381, 387-424, 427-433,435-470, 638-685, 905, 908, or an ECD domain that has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to anyof SEQ ID NOS: 109-142, 239, 280-325, 364-381, 387-424, 427-433,435-470, 638-685, 905, 908 and contains the one or more amino acidmodifications of the respective SEQ ID NO. In particular cases, such animmunomodulatory protein contains a variant ICOSL polypeptide containingan IgV domain, such as an IgV domain set forth in any one of SEQ ID NOS:197-199, 201-208, 210, 212, 240, 326-340, 382-386, 425-426, 434,546-599, 686-857, 906-907, 909-910, or an IgV domain that has at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% to any of SEQ ID NOS: 197-199, 201-208, 210, 212, 240, 326-340,382-386, 425-426, 434, 546-599, 686-857, 906-907, 909-910 and containsthe one or more amino acid modifications of the respective SEQ ID NO. Insome embodiments, the variant ICOSL polypeptide has an IgSF domain (e.g.IgV domain) exhibits increased binding affinity to CD28 or ICOS, such asany described herein. In some embodiments, the variant ICOSL polypeptidehas an IgSF domain (e.g. IgV domain) containing one or more amino acidmodification, e.g. substitution in an reference ICOSL or specificbinding fragment, corresponding to position(s) 52, 57, or 100 withreference to numbering of SEQ ID NO:32. In some embodiments, the variantICOSL polypeptide has one or more amino acid modification, e.g.substitution selected from N52A, N52C, N52D, N52G, N52H, N52L, N52K,N52M, N52P, N52Q, N52R, N52S, N52T, N52V, N52Y, N57A, N57E, N57F, N57H,N57K, N57L, N57M, N57P, N57Q, N57S, N57T, N57V, N57W, N57Y, Q100A,Q100D, Q100E, Q100G, Q100K, Q100L, Q100M, Q100N, Q100R, Q100P, Q100S,Q100T, or Q100V. Exemplary of such variant molecules include any asdescribed herein. In some embodiments, the variant ICOSL polypeptidecontains the amino acid modifications N52D (e.g. is or includes an IgVdomain set forth in SEQ ID NO:548), N52H/Q100R (e.g. is or includes anIgV domain set forth in SEQ ID NO:567), N52H/N57Y/Q100R (e.g. is orincludes an IgV domain set forth in SEQ ID NO:565), or N52L/N57H/Q100R(e.g. is or includes an IgV domain set forth in SEQ ID NO:761).

In some embodiments, the provided multi-domain stack immunomodulatoryproteins, such as an ICOSL-NKp30 multi-domain stack immunomodulatoryprotein, are fused to an Fc polypeptide. In particular embodiments, theFc polypeptide is a variant of a human IgG1 Fc region that exhibitsreduced effector functions, such as any as described. In someembodiments, the Fc region is a human IgG1 that contains the amino acidmodifications N297G, E233P/L234V/L235A/G236del/S267K orL234A/L235E/G237A, wherein the residue is numbered according to the EUindex of Kabat. In some embodiments, the variant IgG1 Fc region furthercontains the amino acid substitution C220S, wherein the residues arenumbered according to the EU index of Kabat. In some embodiments, the Fcregion contains K447del, wherein the residue is numbered according tothe EU index of Kabat. In some aspects, the Fc region contains thesequence of amino acid sequence set forth in any of SEQ ID NOS: 474,476, 477, 478, 633 or 637 or a sequence of amino acids that exhibits atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to any of SEQ ID NOS: 474, 476, 477, 478, 633 or 637 andcontains the amino acid substitutions of the respective SEQ ID NO.

Exemplary of such configurations are set forth in FIGS. 16A-16B anddescribed herein. In some embodiments, any of the provided ICOSL-NKp30multi-domain stack immunomodulatory polypeptides can contain two copiesof a polypeptide having the structure: variant ICOSL IgSF (e.g. IgV,such as set forth in SEQ ID NO: 548, 565, 567 or 761)—linker 1—variantNKp30 IgSF (e.g. IgV, such as set forth in SEQ ID NO: 504)—linker 2—Fc.In some embodiments, any of the provided ICOSL-NKp30 multi-domain stackimmunomodulatory polypeptides can contain two copies of a polypeptidehaving the structure: variant ICOSL IgSF (e.g. IgV, such as set forth inSEQ ID NO: 548, 565, 567 or 761)—linker 1—variant NKp30 IgSF (e.g. IgV,such as set forth in SEQ ID NO:504)—linker 1—variant NKp30 IgSF (e.g.IgV, such as set forth in SEQ ID NO:504)—linker 2—Fc. In someembodiments, linker 1 and linker 2 are peptide linkers, such as any asdescribed. In some embodiments, linker 1 and linker 2 are the same. Insome embodiments, linker 1 and linker 2 are different. In someembodiments, linker 1 is 3×GGGGS (SEQ ID NO: 228). In some embodiments,linker 2 is GSGGGS (SEQ ID NO: 635).

Exemplary ICOSL-NKp30 multidomain stacks have the sequence of aminoacids set forth in any of SEQ ID NOS: 912, 914, 916, 918, 920, 922, 924or 926, or a sequence of amino acids that exhibits at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to anyof SEQ ID NOS: 912, 914, 916, 918, 920, 922, 924 or 926.

In some embodiments, any of the provided ICOSL-NKp30 multi-domain stackimmunomodulatory proteins bind to ICOS and/or CD28 and bind to B7-H6. Insome embodiments, the provided ICOSL-NKp30 multi-domain stackimmunomodulatory proteins provide for a binding molecule capable oftumor localization adjacent to an immune cell that expresses ICOS and/orCD28 (e.g. a T cell). In some embodiments, such ICOSL-NKp30 multi-domainstack immunomodulatory proteins can be used to increase an immuneresponse by engagement of ICOS and/or CD28 costimulatory receptors on Tcells in a tumor microenvironment. In some cases, such ICOSL-NKp30multi-domain stack immunomodulatory proteins, or pharmaceuticalcompositions thereof, can be used to treat a tumor or cancer.

In some embodiments, the multi-domain stack molecule is heterodimeric,comprising two different Fc fusion polypeptides, e.g. a first and asecond Fc polypeptide, wherein at least one is an Fc fusion polypeptidecontaining at least one variant ICOSL polypeptide and/or at least one isan Fc polypeptide containing a second IgSF domain (e.g. second variantIgSF domain). In some embodiments, the first or second Fc fusionpolypeptide further contains a third IgSF domain (e.g. third variantIgSF domain). In some embodiments, the multi-domain stack moleculecontains a first Fc fusion polypeptide containing a variant ICOSL and asecond Fc fusion polypeptide containing at a second IgSF domain, inwhich, in some cases, the first or second Fc fusion polypeptideadditionally contains a third IgSF domain. In some embodiments, themulti-domain stack molecule contains a first Fc fusion polypeptidecontaining a variant ICOSL, a second IgSF domain, and in some cases, athird IgSF domain and a second Fc fusion polypeptide that is not linkedto either a variant ICOSL polypeptide or an additional IgSF domain. Insome embodiments, the Fc portion or region of the first and secondfusion polypeptide is the same. In some embodiments, the Fc portion orregion of the first and second fusion polypeptide is different. In someembodiments, the multi-domain stack molecule contains a first fusion Fcpolypeptide containing 1, 2, 3, 4 or more variant ICOSL polypeptidesand/or 1, 2, 3, 4 or more additional IgSF domains, wherein the totalnumber of IgSF domains in the first stack Fc fusion polypeptide isgreater than 2, 3, 4, 5, 6 or more. In one example of such anembodiment, the second stack Fc fusion polypeptide contains 1, 2, 3, 4or more variant ICOSL polypeptides and/or 1, 2, 3, 4 or more second IgSFdomains, wherein the total number of IgSF domains in the second stack Fcfusion polypeptide is greater than 2, 3, 4, 5, 6 or more. In anotherexample of such an embodiment, the second Fc fusion polypeptide is notlinked to either a variant ICOSL polypeptide or additional IgSF domain.

In some embodiments, the heterodimeric stack molecule contains a firststack immunomodulatory Fc fusion polypeptide and a second stackimmunomodulatory Fc fusion polypeptide in which the first and secondpolypeptide are different. In some embodiments, a heterodimeric stackmolecule contains a first Fc polypeptide fusion containing an Fc regionand a first variant ICOSL polypeptide and/or second IgSF domain (e.g.second variant IgSF domain) and a second Fc polypeptide fusioncontaining an Fc region and the other of the first variant ICOSLpolypeptide or the second IgSF domain. In some embodiments, aheterodimeric stack molecule contains a first Fc polypeptide fusioncontaining an Fc region and a first variant ICOSL polypeptide and/orsecond IgSF domain (e.g. second variant IgSF domain) and a second Fcpolypeptide fusion containing both the first variant ICOSL polypeptideand second IgSF domain (e.g. second variant IgSF domain) but in adifferent orientation or configuration from the first Fc region. In someembodiments, the first and/or second Fc fusion polypeptide also containsa third IgSF domain (e.g. third variant IgSF domain).

In some embodiments, the Fc domain of one or both of the first andsecond stacked immunomodulatory Fc fusion polypeptide comprises amodification (e.g. substitution) such that the interface of the Fcmolecule is modified to facilitate and/or promote heterodimerization. Insome embodiments, modifications include introduction of a protuberance(knob) into a first Fc polypeptide and a cavity (hole) into a second Fcpolypeptide such that the protuberance is positionable in the cavity topromote complexing of the first and second Fc-containing polypeptides.Amino acids targeted for replacement and/or modification to createprotuberances or cavities in a polypeptide are typically interface aminoacids that interact or contact with one or more amino acids in theinterface of a second polypeptide.

In some embodiments, a sequence of amino acids is added preceding the Fcsequence for constructs in which the Fc sequence is the N-terminalportion of the sequence. In some cases, the sequence of amino acidsHMSSVSAQ (SEQ ID NO:475) is added immediately preceding the Fc sequencefor constructs in which the Fc sequence is the N-terminal portion of thesequence. In some embodiments, a heterodimeric stack molecule contains afirst Fc polypeptide fusion containing an Fc region (knob) and a firstvariant ICOSL polypeptide and/or second IgSF domain (e.g. second variantIgSF domain) and a second Fc polypeptide fusion containing an Fc region(hole) and a stuffer sequence HMSSVSAQ (SEQ ID NO:475) added immediatelypreceding both Fc regions of the first and second Fc polypeptide fusion.

In some embodiments, a first polypeptide that is modified to containprotuberance (hole) amino acids include replacement of a native ororiginal amino acid with an amino acid that has at least one side chainwhich projects from the interface of the first polypeptide and istherefore positionable in a compensatory cavity (hole) in an adjacentinterface of a second polypeptide. Most often, the replacement aminoacid is one which has a larger side chain volume than the original aminoacid residue. One of skill in the art knows how to determine and/orassess the properties of amino acid residues to identify those that areideal replacement amino acids to create a protuberance. In someembodiments, the replacement residues for the formation of aprotuberance are naturally occurring amino acid residues and include,for example, arginine (R), phenylalanine (F), tyrosine (Y), ortryptophan (W). In some examples, the original residue identified forreplacement is an amino acid residue that has a small side chain suchas, for example, alanine, asparagine, aspartic acid, glycine, serine,threonine, or valine.

In some embodiments, a second polypeptide that is modified to contain acavity (hole) is one that includes replacement of a native or originalamino acid with an amino acid that has at least one side chain that isrecessed from the interface of the second polypeptide and thus is ableto accommodate a corresponding protuberance from the interface of afirst polypeptide. Most often, the replacement amino acid is one whichhas a smaller side chain volume than the original amino acid residue.One of skill in the art knows how to determine and/or assess theproperties of amino acid residues to identify those that are idealreplacement residues for the formation of a cavity. Generally, thereplacement residues for the formation of a cavity are naturallyoccurring amino acids and include, for example, alanine (A), serine (S),threonine (T) and valine (V). In some examples, the original amino acididentified for replacement is an amino acid that has a large side chainsuch as, for example, tyrosine, arginine, phenylalanine, or typtophan.

The CH3 interface of human IgG1, for example, involves sixteen residueson each domain located on four anti-parallel β-strands which buries 1090Å2 from each surface (see e.g., Deisenhofer et al. (1981) Biochemistry,20:2361-2370; Miller et al., (1990) J Mol. Biol., 216, 965-973; Ridgwayet al., (1996) Prot. Engin., 9: 617-621; U.S. Pat. No. 5,731,168).Modifications of a CH3 domain to create protuberances or cavities aredescribed, for example, in U.S. Pat. No. 5,731,168; International PatentApplications WO98/50431 and WO 2005/063816; and Ridgway et al., (1996)Prot. Engin., 9: 617-621. In some examples, modifications of a CH3domain to create protuberances or cavities are typically targeted toresidues located on the two central anti-parallel β-strands. The aim isto minimize the risk that the protuberances which are created can beaccommodated by protruding into the surrounding solvent rather thanbeing accommodated by a compensatory cavity in the partner CH3 domain.

In some embodiments, the heterodimeric molecule contains a T366Wmutation in the CH3 domain of the “knobs chain” and T366S, L368A, Y407Vmutations in the CH3 domain of the “hole chain”. In some cases, anadditional interchain disulfide bridge between the CH3 domains can alsobe used (Merchant, A. M., et al., Nature Biotech. 16 (1998) 677-681)e.g. by introducing a Y349C mutation into the CH3 domain of the “knobs”or “hole” chain and a E356C mutation or a S354C mutation into the CH3domain of the other chain. In some embodiments, the heterodimericmolecule contains S354C, T366W mutations in one of the two CH3 domainsand Y349C, T366S, L368A, Y407V mutations in the other of the two CH3domains. In some embodiments, the heterodimeric molecule comprisesE356C, T366W mutations in one of the two CH3 domains and Y349C, T366S,L368A, Y407V mutations in the other of the two CH3 domains. In someembodiments, the heterodimeric molecule comprises Y349C, T366W mutationsin one of the two CH3 domains and E356C, T366S, L368A, Y407V mutationsin the other of the two CH3 domains. In some embodiments, theheterodimeric molecule comprises Y349C, T366W mutations in one of thetwo CH3 domains and S354C, T366S, L368A, Y407V mutations in the other ofthe two CH3 domains. Examples of other knobs-in-holes technologies areknown in the art, e.g. as described by EP 1 870 459 A1.

In some embodiments, the Fc regions of the heterodimeric moleculeadditionally can contain one or more other Fc mutation, such as anydescribed above. In some embodiments, the heterodimer molecule containsan Fc region with a mutation that reduces effector function.

In some embodiments, an Fc variant containing CH3 protuberance (knob) orcavity (hole) modifications can be joined to a stacked immunomodulatorypolypeptide anywhere, but typically via its N- or C-terminus, to the N-or C-terminus of a first and/or second stacked immunomodulatorypolypeptide, such as to form a fusion polypeptide. The linkage can bedirect or indirect via a linker. Typically, a knob and hole molecule isgenerated by co-expression of a first stacked immunomodulatorypolypeptide linked to an Fc variant containing CH3 protuberancemodification(s) with a second stacked immunomodulatory polypeptidelinked to an Fc variant containing CH3 cavity modification(s).

Also provided are nucleic acid molecules encoding the variant ICOSL-Fcfusion protein. In some embodiments, for production of an Fc fusionprotein, a nucleic acid molecule encoding a variant ICOSL-Fc fusionprotein is inserted into an appropriate expression vector. The resultingvariant ICOSL-Fc fusion protein can be expressed in host cellstransformed with the expression where assembly between Fc domains occursby interchain disulfide bonds formed between the Fc moieties to yielddimeric, such as divalent, variant ICOSL-Fc fusion proteins.

The resulting Fc fusion proteins can be easily purified by affinitychromatography over Protein A or Protein G columns. For the generationof heterodimers, additional steps for purification can be necessary. Forexample, where two nucleic acids encoding different variant ICOSLpolypeptides are transformed into cells, the formation of heterodimersmust be biochemically achieved since variant ICOSL molecules carryingthe Fc-domain will be expressed as disulfide-linked homodimers as well.Thus, homodimers can be reduced under conditions that favor thedisruption of interchain disulfides, but do no effect intra-chaindisulfides. In some cases, different variant-ICOSL Fc monomers are mixedin equimolar amounts and oxidized to form a mixture of homo- andheterodimers. The components of this mixture are separated bychromatographic techniques. Alternatively, the formation of this type ofheterodimer can be biased by genetically engineering and expressing Fcfusion molecules that contain a variant ICOSL polypeptide usingknob-into-hole methods described below.

B. Conjugates and Fusions of Variant Polypeptides and ImmunomodulatoryProteins

In some embodiments, the variant polypeptides provided herein, which areimmunomodulatory proteins comprising variants of an Ig domain of theIgSF family (vIgD), can be conjugated with, such as fused directly orindirectly, to, a moiety, such as an effector moiety, such as anotherprotein, directly or indirectly, to form a conjugate (“IgSF conjugate”).In some embodiments, a variant ICOSL immunomodulatory protein isprovided as a conjugate in which is contained a vIgD of ICOSL linked,directly or indirectly, to a targeting agent or moiety, e.g. to anantibody or other binding molecules that specifically binds to a ligand,e.g. an antigen, for example, for targeting or localizing the vIgD to aspecific environment or cell, such as when administered to a subject. Insome embodiments, the targeting agent, e.g. antibody or other bindingmolecule, binds to a tumor antigen, thereby localizing the variant ICOSLcontaining the vIgD to the tumor microenvironment, for example, tomodulate activity of tumor infiltrating lymphocytes (TILs) specific tothe tumor microenvironment. In some embodiments, the attachment can becovalent or non-covalent, e.g., via a biotin-streptavidin non-covalentinteraction. In some embodiments, the conjugate is a fusion protein of avariant ICOSL polypeptide linked, directly or via a linker, to anotherprotein or polypeptide moiety.

In some embodiments the fusion protein is an ICOSL-Fc variant fusion, inwhich any two or more of the foregoing variant polypeptides can beattached to an Fc.

In some embodiments, the IgSF conjugate, such as fusion protein,comprises the ECD of a wildtype (full length or truncated) or a variantICOSL polypeptide. In some embodiments, the IgSF conjugate, such asfusion protein, comprises an IgV domain or an IgC (e.g., IgC2) domain ordomains, or a specific binding fragment of the IgV domain or a specificbinding fragment of the IgC (e.g., IgC2) domain or domains. In someembodiments, the IgSF conjugate, such as fusion protein, comprises anIgV domain of ICOSL as set forth in SEQ ID NOs: 196 or 545.

In some embodiments, the moiety can be a targeting moiety, a smallmolecule drug (non-polypeptide drug of less than 500 daltons molarmass), a toxin, a cytostatic agent, a cytotoxic agent, animmunosuppressive agent, a radioactive agent suitable for diagnosticpurposes, a radioactive metal ion for therapeutic purposes, aprodrug-activating enzyme, an agent that increases biological half-life,or a diagnostic or detectable agent.

In some embodiments the effector moiety is a therapeutic agent, such asa cancer therapeutic agent, which is either cytotoxic, cytostatic orotherwise provides some therapeutic benefit. In some embodiments, theeffector moiety is a targeting moiety or agent, such as an agent thattargets a cell surface antigen, e.g., an antigen on the surface of atumor cell. In some embodiments, the effector moiety is a label, whichcan generate a detectable signal, either directly or indirectly. In someembodiments, the effector moiety is a toxin. In some embodiments, theeffector moiety is a protein, peptide, nucleic acid, small molecule ornanoparticle.

In some embodiments, 1, 2, 3, 4, 5 or more effector moieties, which canbe the same or different, are conjugated, linked or fused to the variantpolypeptide or protein to form an IgSF conjugate. In some embodiments,such effector moieties can be attached to the variant polypeptide orimmunomodulatory protein using various molecular biological or chemicalconjugation and linkage methods known in the art and described below. Insome embodiments, linkers such as peptide linkers, cleavable linkers,non-cleavable linkers or linkers that aid in the conjugation reaction,can be used to link or conjugate the effector moieties to the variantpolypeptide or immunomodulatory protein.

In some embodiments, the IgSF conjugate comprises the followingcomponents: (protein or polypeptide), (L)_(q) and (effector moiety)_(m),wherein the protein or polypeptide is any of the described variantpolypeptides or immunomodulatory proteins capable of binding one or morecognate counter structure ligands as described; L is a linker forlinking the protein or polypeptide to the moiety; m is at least 1; q is0 or more; and the resulting IgSF conjugate binds to the one or morecounter structure ligands. In particular embodiments, m is 1 to 4 and qis 0 to 8. In some embodiments, the linker is a peptide. In someembodiments, the effector moiety is a protein or polypeptide.

In some embodiments, there is provided an IgSF conjugate comprising avariant polypeptide or immunomodulatory protein provided hereinconjugated with a targeting agent that binds to a cell surface molecule,for example, for targeted delivery of the variant polypeptide orimmunomodulatory protein to a specific cell. In some embodiments, thetargeting agent is a molecule(s) that has the ability to localize andbind to a molecule present on a normal cell/tissue and/or tumorcell/tumor in a subject. In other words, IgSF conjugates comprising atargeting agent can bind to a ligand (directly or indirectly), which ispresent on a cell, such as a tumor cell. The targeting agents of theinvention contemplated for use include antibodies, polypeptides,peptides, aptamers, other ligands, or any combination thereof, that canbind a component of a target cell or molecule.

In some embodiments, the targeting agent binds a tumor cell(s) or canbind in the vicinity of a tumor cell(s) (e.g., tumor vasculature ortumor microenvironment) following administration to the subject. Thetargeting agent may bind to a receptor or ligand on the surface of thecancer cell. In another aspect of the invention, a targeting agent isselected which is specific for a noncancerous cells or tissue. Forexample, a targeting agent can be specific for a molecule presentnormally on a particular cell or tissue. Furthermore, in someembodiments, the same molecule can be present on normal and cancercells. Various cellular components and molecules are known. For example,if a targeting agent is specific for EGFR, the resulting IgSF conjugatecan target cancer cells expressing EGFR as well as normal skin epidermalcells expressing EGFR. Therefore, in some embodiments, an IgSF conjugateof the invention can operate by two separate mechanisms (targetingcancer and non-cancer cells).

In various aspects of the invention disclosed herein an IgSF conjugateof the invention comprises a targeting agent which can bind/target acellular component, such as a tumor antigen, a bacterial antigen, aviral antigen, a mycoplasm antigen, a fungal antigen, a prion antigen,an antigen from a parasite. In some aspects, a cellular component,antigen or molecule can each be used to mean, a desired target for atargeting agent. For example, in various embodiments, a targeting agentis specific for or binds to a component, which includes but is notlimited to, epidermal growth factor receptor (EGFR, ErbB-1, HER1),ErbB-2 (HER2/neu), ErbB-3/HER3, ErbB-4/HER4, EGFR ligand family;insulin-like growth factor receptor (IGFR) family, IGF-binding proteins(IGFBPs), IGFR ligand family; platelet derived growth factor receptor(PDGFR) family, PDGFR ligand family; fibroblast growth factor receptor(FGFR) family, FGFR ligand family, vascular endothelial growth factorreceptor (VEGFR) family, VEGF family; HGF receptor family; TRK receptorfamily; ephrin (EPH) receptor family; AXL receptor family; leukocytetyrosine kinase (LTK) receptor family; TIE receptor family, angiopoietin1,2; receptor tyrosine kinase-like orphan receptor (ROR) receptorfamily, e.g. ROR1; CD171 (L1CAM); B7-H6 (NCR3LG1); PD-L1, tumorglycosylation antigen, e.g. sTn or Tn, such as sTn Ag of MUC1; LHR(LHCGR); phosphatidylserine, discoidin domain receptor (DDR) family; RETreceptor family; KLG receptor family; RYK receptor family; MuSK receptorfamily; Transforming growth factor-α (TGF-α) receptors, TGF-β; Cytokinereceptors, Class I (hematopoietin family) and Class II (interferon/IL-10family) receptors, tumor necrosis factor (TNF) receptor superfamily(TNFRSF), death receptor family; cancer-testis (CT) antigens,lineage-specific antigens, differentiation antigens, alpha-actinin-4,ARTC1, breakpoint cluster region-Abelson (Bcr-abl) fusion products,B-RAF, caspase-5 (CASP-5), caspase-8 (CASP-8), β-catenin (CTNNB1), celldivision cycle 27 (CDC27), cyclin-dependent kinase 4 (CDK4), CDKN2A,COA-I, dek-can fusion protein, EFTUD-2, Elongation factor 2 (ELF2), Etsvariant gene 6/acute myeloid leukemia 1 gene ETS (ETC6-AML1) fusionprotein, fibronectin (FN), e.g. the extradomain A (EDA) of fibronectin,GPNMB, low density lipid receptor/GDP-L fucose: 13-D-galactose2-α-L-fucosyltransferase (LDLR/FUT) fusion protein, HLA-A2. arginine toisoleucine exchange at residue 170 of the α-helix of the α2-domain inthe HLA-A2gene (HLA-A*201-R170I), HLA-AI 1, heat shock protein 70-2mutated (HSP70-2M), K1AA0205, MART2, melanoma ubiquitous mutated 1, 2, 3(MUM-I, 2, 3), prostatic acid phosphatase (PAP), neo-PAP, Myosin classI, NFYC, OGT, OS-9, pml-RARα fusion protein, PRDX5, PTPRK, K-ras(KRAS2), N-ras (NRAS), HRAS, RBAF600, SIRT2, SNRPD1, SYT-SSX1 or -SSX2fusion protein, Triosephosphate Isomerase, BAGE, BAGK-1, BAGE-2,3,4,5,GAGE-1,2,3,4,5,6,7,8, GnT-V (aberrant N-acetyl glucosaminyl transferaseV, MGAT5), HERV-K-MEL, KK-LC, KM-HN-I, LAGE, LAGE-I, CTL-recognizedantigen on melanoma (CAMEL), MAGE-A1 (MAGE-I), MAGE-A2, MAGE-A3,MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A1O, MAGE-AI1,MAGE-A12, MAGE-3, MAGE-B1, MAGE-B2, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2,mucin 1 (MUC1), MART-1/Melan-A (MLANA), gp100, gp100/Pmell7 (SILV),tyrosinase (TYR), TRP-I, HAGE, NA-88, NY-ESO-I, NY-ESO-1/LAGE-2, SAGE,Sp17, SSX-1,2,3,4, TRP2-INT2, carcino-embryonic antigen (CEA),Kallikrein 4, mammaglobin-A, OA1, prostate specific antigen (PSA),TRP-1/gp75, TRP-2, adipophilin, interferon inducible protein absent inmelanoma 2 (AIM-2), BING-4, CPSF, cyclin D1, epithelial cell adhesionmolecule (Ep-CAM), EphA3, fibroblast growth factor-5 (FGF-5),glycoprotein 250 (gp250), EGFR (ERBB1), HER-2/neu (ERBB2), interleukin13 receptor α2 chain (IL13Rα2), IL-6 receptor, intestinal carboxylesterase (iCE), alpha-feto protein (AFP), M-CSF, mdm-2, MUC1, p53(TP53), PBF, PRAME, PSMA, RAGE-I, RNF43, RU2AS, SOX1O, STEAP1, survivin(BIRC5), human telomerase reverse transcriptase (hTERT), telomerase,Wilms' tumor gene (WT1), SYCP1, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIP1,CTAGE-I, CSAGE, MMA1, CAGE, BORIS, HOM-TES-85, AF15q14, HCA661, LDHC,MORC, SGY-I, SPO1 1, TPX1, NY-SAR-35, FTHL17, NXF2, TDRD1, TEX15, FATE,TPTE, immunoglobulin idiotypes, Bence-Jones protein, estrogen receptors(ER), androgen receptors (AR), CD40, CD30, CD20, CD19, CD33, cancerantigen 72-4 (CA 72-4), cancer antigen 15-3 (CA 15-3), cancer antigen27-29 (CA 27-29), cancer antigen 125 (CA 125), cancer antigen 19-9 (CA19-9), β-human chorionic gonadotropin, 0-2 microglobulin, squamous cellcarcinoma antigen, neuron-specific enolase, heat shock protein gp96,GM2, sargramostim, CTLA-4, 707 alanine proline (707-AP), adenocarcinomaantigen recognized by T cells 4 (ART-4), carcinoembryogenic antigenpeptide-1 (CAP-I), calcium-activated chloride channel-2 (CLCA2),cyclophilin B (Cyp-B), human signet ring tumor-2 (HST-2), Humanpapilloma virus (HPV) proteins (HPV-E6, HPV-E7, major or minor capsidantigens, others), Epstein-Barr virus (EBV) proteins (EBV latentmembrane proteins—LMP1, LMP2; others), Hepatitis B or C virus proteins,and HIV proteins.

In some embodiments, a targeting agent is specific for or binds to acomponent, which includes, but is not limited to, HER1/EGFR, HER2/ERBB2,CD20, CD25 (IL-2Rα receptor), CD33, CD52, CD133, CD206, CEA, CEACAM1,CEACAM3, CEACAM5, CEACAM6, cancer antigen 125 (CA125), alpha-fetoprotein(AFP), Lewis Y, TAG72, Caprin-1, mesothelin, PDGF receptor (PDGFR; suchas PDGF-R α), PD-1, PD-L1, CTLA-4, IL-2 receptor, vascular endothelialgrowth factor (VEGF), CD30, EpCAM, EphA2, Glypican-3, gpA33, mucins,CAIX, PSMA, folate-binding protein, gangliosides (such as GD2, GD3, GM1and GM2), VEGF receptor (VEGFR), VEGFR2, VEGF-A, integrin αVβ3, integrinα5β1, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin,AFP, BCR complex, CD3, CD18, CD44, CTLA-4, gp72, HLA-DR 10β, HLA-DRantigen, IgE, MUC-1, nuC242, PEM antigen, metalloproteinases, Ephrinreceptor, Ephrin ligands, HGF receptor, CXCR4, CXCR4, Bombesin receptor,SK-1antigen, Bcr-abl, RET, MET, TRKB, TIE2, ALK, ROS, EML4-ALK, ROS1,BRAFV600E, SRC, c-KIT, mTOR, TSC1, TSC2, BTK, KIT, BRCA, CDK 4/6, JAK1,JAK2, BRAF, FLT-3, MEK1, MEK2, SMO or B7-H6 (NCR3LG1).

In some embodiments, an IgSF conjugate, through its targeting agent,will bind a cellular component of a tumor cell, tumor vasculature ortumor microenvironment, thereby promoting killing of targeted cells viamodulation of the immune response, (e.g., by activation ofco-stimulatory molecules or inhibition of negative regulatory moleculesof immune cell activation), inhibition of survival signals (e.g., growthfactor or cytokine or hormone receptor antagonists), activation of deathsignals, and/or immune-mediated cytotoxicity, such as through antibodydependent cellular cytotoxicity. Such IgSF conjugates can functionthrough several mechanisms to prevent, reduce or eliminate tumor cells,such as to facilitate delivery of conjugated effector moieties to thetumor target, such as through receptor-mediated endocytosis of the IgSFconjugate; or such conjugates can recruit, bind, and/or activate immunecells (e.g. NK cells, monocytes/macrophages, dendritic cells, T cells, Bcells). Moreover, in some instances one or more of the foregoingpathways may operate upon administration of one or more IgSF conjugatesof the invention.

In some embodiments, an IgSF conjugate, through its targeting agent,will be localized to, such as bind to, a cellular component of a tumorcell, tumor vasculature or tumor microenvironment, thereby modulatingcells of the immune response in the vicinity of the tumor. In someembodiments, the targeting agent facilitates delivery of the conjugatedIgSF (e.g. vIgD) to the tumor target, such as to interact with itscognate binding partner to alter signaling of immune cells (e.g. NKcells, monocytes/macrophages, dendritic cells, T cells, B cells) bearingthe cognate binding partner. In some embodiments, localized deliveryagonizes or stimulates the costimulatory receptor.

In some embodiments, the targeting agent is an immunoglobulin. As usedherein, the term “immunoglobulin” includes natural or artificial mono-or polyvalent antibodies including, but not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, single chain Fv (scFv);anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto antibodies of the invention), and epitope-binding fragments of any ofthe above. The term “antibody,” as used herein, refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, e.g., molecules that contain an antigen binding site thatimmunospecifically binds an antigen. The immunoglobulin molecules of theinvention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass ofimmunoglobulin molecule.

In some embodiments, an IgSF conjugate, through its antibody targetingmoiety, will bind a cellular component of a tumor cell, tumorvasculature or tumor microenvironment, thereby promoting apoptosis oftargeted cells via modulation of the immune response, (e.g., byactivation of co-stimulatory molecules or inhibition of negativeregulatory molecules of immune cell activation), inhibition of survivalsignals (e.g., growth factor or cytokine or hormone receptorantagonists), activation of death signals, and/or immune-mediatedcytotoxicity, such as through antibody dependent cellular cytotoxicity.Such IgSF conjugates can function through several mechanisms to prevent,reduce or eliminate tumor cells, such as to facilitate delivery ofconjugated effector moieties to the tumor target, such as throughreceptor-mediated endocytosis of the IgSF conjugate; or such conjugatescan recruit, bind, and/or activate immune cells (e.g. NK cells,monocytes/macrophages, dendritic cells, T cells, B cells).

In some embodiments, an IgSF conjugate, through its antibody targetingmoiety, will bind a cellular component of a tumor cell, tumorvasculature or tumor microenvironment, thereby modulating the immuneresponse (e.g., by activation of co-stimulatory molecules or inhibitionof negative regulatory molecules of immune cell activation). In someembodiments, such conjugates can recognize, bind, and/or modulate (e.g.inhibit or activate) immune cells (e.g. NK cells, monocytes/macrophages,dendritic cells, T cells, B cells).

Antibody targeting moieties of the invention include antibody fragmentsthat include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv) and fragments comprising either a VL or VH domain.Antigen-binding antibody fragments, including single-chain antibodies,may comprise the variable region(s) alone or in combination with theentirety or a portion of the following: hinge region, CH1, CH2, and CH3domains. Also included in the invention are antigen-binding fragmentsalso comprising any combination of variable region(s) with a hingeregion, CH1, CH2, and CH3 domains. Also included in the invention are Fcfragments, antigen-Fc fusion proteins, and Fc-targeting moietyconjugates or fusion products (Fc-peptide, Fc-aptamer). The antibodytargeting moieties of the invention may be from any animal originincluding birds and mammals. In one aspect, the antibody targetingmoieties are human, murine (e.g., mouse and rat), donkey, sheep, rabbit,goat, guinea pig, camel, horse, or chicken. Further, such antibodies maybe humanized versions of animal antibodies. The antibody targetingmoieties of the invention may be monospecific, bispecific, trispecific,or of greater multispecificity.

In various embodiments, an antibody/targeting moiety recruits, binds,and/or activates immune cells (e.g. NK cells, monocytes/macrophages,dendritic cells) via interactions between Fc (in antibodies) and Fcreceptors (on immune cells) and via the conjugated variant polypeptidesor immunomodulatory proteins provided herein. In some embodiments, anantibody/targeting moiety recognizes or binds a tumor agent via andlocalizes to the tumor cell the conjugated variant polypeptides orimmunomodulatory proteins provided herein to facilitate modulation ofimmune cells in the vicinity of the tumor.

Examples of antibodies which can be incorporated into IgSF conjugatesinclude but are not limited to antibodies such as Cetuximab (IMC-C225;Erbitux®), Trastuzumab (Herceptin®), Rituximab (Rituxan®; MabThera®),Bevacizumab (Avastin®), Alemtuzumab (Campath®; Campath-1H®;Mabcampath®), Pertuzumab (Perjeta®), Panitumumab (ABX-EGF; Vectibix®),Ranibizumab (Lucentis®), Ibritumomab, Ibritumomab tiuxetan, (Zevalin®),Tositumomab, Iodine I131 Tositumomab (BEXXAR®), Catumaxomab (Removab®),Dinutuximab (Unituxin™), Gemtuzumab, Gemtuzumab ozogamicine (Mylotarg®),Abatacept (CTLA4-Ig; Orencia®), Belatacept (L104EA29YIg; LEA29Y; LEA),Ipilimumab (MDX-010; MDX-101), Tremelimumab (ticilimumab; CP-675,206),PRS-010, PRS-050, Aflibercept (VEGF Trap, AVE005), Volociximab (M200),F200, MORAb-009, SS1P (CAT-5001), Cixutumumab (IMC-A12), Matuzumab(EMD72000), Nimotuzumab (h-R3), Zalutumumab (HuMax-EGFR), NecitumumabIMC-11F8, mAb806/ch806, Sym004, mAb-425, Panorex @ (17-1A) (murinemonoclonal antibody); Panorex @ (17-1A) (chimeric murine monoclonalantibody); IDEC-Y2B8 (murine, anti-CD20 MAb); BEC2 (anti-idiotypic MAb,mimics the GD epitope) (with BCG); Olaratumab (Lartruvo™); Oncolym(Lym-1 monoclonal antibody); SMART MI95 Ab, humanized 13′ I LYM-I(Oncolym), Ovarex (B43.13, anti-idiotypic mouse MAb); Ramucirumab(Cyramza®); MDX-210 (humanized anti-HER-2 bispecific antibody); 3622W94MAb that binds to EGP40 (17-1A) pancarcinoma antigen on adenocarcinomas;Anti-VEGF, Zenapax (SMART Anti-Tac (IL-2 receptor); SMART MI95 Ab,humanized Ab, humanized); MDX-210 (humanized anti-HER-2 bispecificantibody); MDX-447 (humanized anti-EGF receptor bispecific antibody);NovoMAb-G2 (pancarcinoma specific Ab); TNT (chimeric MAb to histoneantigens); TNT (chimeric MAb to histone antigens); Gliomab-H (Monoclons—Humanized Abs); GNI-250 Mab; EMD-72000 (chimeric-EGF antagonist);LymphoCide (humanized LL2 antibody); and MDX-260 bispecific, targetsGD-2, ANA Ab, SMART ID1O Ab, SMART ABL 364 Ab or ImmuRAIT-CEA. Asillustrated by the forgoing list, it is conventional to make antibodiesto a particular target epitope. In some embodiments, the antibody orantigen-binding fragment of the provided conjugates, including fusionmolecules, is cetuximab, panitumumab, zalutumumab, nimotuzumab,trastuzumab, Ado-trastuzumab emtansine, Tositumomab (Bexxar®), Rituximab(Rituxan, Mabthera), Ibritumomab tiuxetan (Zevalin), Daclizumab(Zenapax), Gemtuzumab (Mylotarg), Alemtuzumab, CEA-scan Fab fragment,OC125 monoclonal antibody, ab75705, B72.3, Bevacizumab (Avastin®),Afatinib, Axitinib, Bosutinib, Cabozantinib, Ceritinib, Crizotinib,Dabrafenib, Dasatinib, Dinutuximab (Unituxin™), Erlotinib, Everolimus,Ibrutinib, Imatinib, Lapatinib, Lenvatinib, Nilotinib, Olaparib,Olaratumab (Lartruvo™), Palbociclib, Pazopanib, Pertuzumab (Perjeta®),Ramucirumab (Cyramza®), Regorafenib, Ruxolitinib, Sorafenib, Sunitinib,Temsirolimus, Trametinib, Vandetanib, Vemurafenib, Vismodegib,Basiliximab, Ipilimumab, Nivolumab, pembrolizumab, MPDL3280A,Pidilizumab (CT-011), AMP-224, MSB001078C, or MEDI4736, BMS-935559,LY3300054, atezolizumab, avelumab or durvalumab or is an antigen-bindingfragment thereof.

In some embodiments, PD-L1 antibodies or antigen binding fragmentsthereof can be incorporated into the IgSF conjugates. Examples of PD-L1antibodies which can be incorporated into IgSF conjugates include butare not limited to antibodies such as BMS-936559, 12A4, LY3300054,Atezolizumab (Tecentriq®), Avelumab (Bavencio®), Durvalumab (Imfinzi®).See, e.g., WO2007/005874, WO2017/034916, WO2010/077634, WO2013/079174,WO2011/066389, these references are incorporated by reference in theirentirety. In some embodiments, the vIgD is linked, directly orindirectly, to the N- or C-terminus of the light and/or heavy chain ofan anti-PD-L1 antibody. In some embodiments, the anti-PD-L antibody isBMS-936559, LY3300054, atezolizumab, avelumab or durvalumab. Exemplarylight chain and heavy chain of an anti-PD-L1 antibody atezolizumab areset forth in SEQ ID NO: 866 and 867, respectively. Exemplary IgSFconjugates that include the anti-PD-L1 antibody Atezolizumab is setforth in SEQ ID NOs: 868-895.

In some embodiments, the antibody targeting moiety is a full lengthantibody, or antigen-binding fragment thereof, containing an Fc domain.In some embodiments, the variant polypeptide or immunomodulatory proteinis conjugated to the Fc portion of the antibody targeting moiety, suchas by conjugation to the N-terminus of the Fc portion of the antibody.

In some embodiments, the vIgD is linked, directly or indirectly, to theN- or C-terminus of the light and/or heavy chain of the antibody. Insome embodiments, linkage can be via a peptide linker, such as anydescribed above. In some embodiments, the linker can further includeamino acids introduced by cloning and/or from a restriction site. Insome embodiments, the linker may include additional amino acids oneither end introduced by a restriction site. For example, the linker caninclude additional amino acids such as SA (in one-letter amino acidcode) as introduced by use of the restriction site AFEI. Variousconfigurations can be constructed. FIG. 10A-10C depict exemplaryconfigurations. In some embodiments, the antibody conjugate can beproduced by co-expression of the heavy and light chain of the antibodyin a cell.

In one aspect of the invention, the targeting agent is an aptamermolecule. For example, in some embodiments, the aptamer is comprised ofnucleic acids that function as a targeting agent. In variousembodiments, an IgSF conjugate of the invention comprises an aptamerthat is specific for a molecule on a tumor cell, tumor vasculature,and/or a tumor microenvironment. In some embodiments, the aptamer itselfcan comprise a biologically active sequence, in addition to thetargeting module (sequence), wherein the biologically active sequencecan induce an immune response to the target cell. In other words, suchan aptamer molecule is a dual use agent. In some embodiments, an IgSFconjugate of the invention comprises conjugation of an aptamer to anantibody, wherein the aptamer and the antibody are specific for bindingto separate molecules on a tumor cell, tumor vasculature, tumormicroenvironment, and/or immune cells.

The term “aptamer” includes DNA, RNA or peptides that are selected basedon specific binding properties to a particular molecule. For example, anaptamer(s) can be selected for binding a particular gene or gene productin a tumor cell, tumor vasculature, tumor microenvironment, and/or animmune cell, as disclosed herein, where selection is made by methodsknown in the art and familiar to one of skill in the art.

In some aspects of the invention the targeting agent is a peptide. Forexample, the variant polypeptides or immunomodulatory proteins providedherein can be conjugated to a peptide which can bind with a component ofa cancer or tumor cells. Therefore, such IgSF conjugates of theinvention comprise peptide targeting agents which binds to a cellularcomponent of a tumor cell, tumor vasculature, and/or a component of atumor microenvironment. In some embodiments, targeting agent peptidescan be an antagonist or agonist of an integrin. Integrins, whichcomprise an alpha and a beta subunit, include numerous types well knownto a skilled artisan.

In one embodiment, the targeting agent is Vvβ3. Integrin Vvβ3 isexpressed on a variety of cells and has been shown to mediate severalbiologically relevant processes, including adhesion of osteoclasts tobone matrix, migration of vascular smooth muscle cells, andangiogenesis. Suitable targeting molecules for integrins include RGDpeptides or peptidomimetics as well as non-RGD peptides orpeptidomimetics (see, e.g., U.S. Pat. Nos. 5,767,071 and 5,780,426) forother integrins such as V4.βi (VLA-4), V4-P7 (see, e.g., U.S. Pat. No.6,365,619; Chang et al, Bioorganic & Medicinal Chem Lett, 12:159-163(2002); Lin et al., Bioorganic & Medicinal Chem Lett, 12:133-136(2002)), and the like.

In some embodiments, there is provided an IgSF conjugate comprising avariant polypeptide or immunomodulatory protein provided hereinconjugated with a therapeutic agent. In some embodiments, thetherapeutic agent includes, for example, daunomycin, doxorubicin,methotrexate, and vindesine (Rowland et al., Cancer Immunol. Immunother.21:183-187, 1986). In some embodiments, the therapeutic agent has anintracellular activity. In some embodiments, the IgSF conjugate isinternalized and the therapeutic agent is a cytotoxin that blocks theprotein synthesis of the cell, therein leading to cell death. In someembodiments, the therapeutic agent is a cytotoxin comprising apolypeptide having ribosome-inactivating activity including, forexample, gelonin, bouganin, saporin, ricin, ricin A chain, bryodin,diphtheria toxin, restrictocin, Pseudomonas exotoxin A and variantsthereof. In some embodiments, where the therapeutic agent is a cytotoxincomprising a polypeptide having a ribosome-inactivating activity, theIgSF conjugate must be internalized upon binding to the target cell inorder for the protein to be cytotoxic to the cells.

In some embodiments, there is provided an IgSF conjugate comprising avariant polypeptide or immunomodulatory protein provided hereinconjugated with a toxin. In some embodiments, the toxin includes, forexample, bacterial toxins such as diphtheria toxin, plant toxins such asricin, small molecule toxins such as geldanamycin (Mandler et al., J.Nat. Cancer Inst. 92(19):1573-1581 (2000); Mandler et al., Bioorganic &Med. Chem. Letters 10: 1025-1028 (2000); Mandler et al., BioconjugateChem. 13:786-791 (2002)), maytansinoids (EP 1391213; Liu et al., Proc.Natl. Acad. Sci. USA 93:8618-8623 (1996)), and calicheamicin (Lode etal., Cancer Res. 58:2928 (1998); Hinman et al., Cancer Res. 53:3336-3342(1993)). The toxins may exert their cytotoxic and cytostatic effects bymechanisms including tubulin binding, DNA binding, or topoisomeraseinhibition.

In some embodiments, there is provided an IgSF conjugate comprising avariant polypeptide or immunomodulatory protein provided hereinconjugated with a label, which can generate a detectable signal,indirectly or directly. These IgSF conjugates can be used for researchor diagnostic applications, such as for the in vivo detection of cancer.The label is preferably capable of producing, either directly orindirectly, a detectable signal. For example, the label may beradio-opaque or a radioisotope, such as 3H, 14C, 32P, 35S, 1231, 1251,131I; a fluorescent (fluorophore) or chemiluminescent (chromophore)compound, such as fluorescein isothiocyanate, rhodamine or luciferin; anenzyme, such as alkaline phosphatase,β-galactosidase or horseradishperoxidase; an imaging agent; or a metal ion. In some embodiments, thelabel is a radioactive atom for scintigraphic studies, for example 99Tcor 1231, or a spin label for nuclear magnetic resonance (NMR) imaging(also known as magnetic resonance imaging, MRI), such as zirconium-89,iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,oxygen-17, gadolinium, manganese or iron. Zirconium-89 may be complexedto various metal chelating agents and conjugated to antibodies, e.g.,for PET imaging (WO 2011/056983). In some embodiments, the IgSFconjugate is detectable indirectly. For example, a secondary antibodythat is specific for the IgSF conjugate and contains a detectable labelcan be used to detect the IgSF conjugate.

The IgSF conjugates may be prepared using any methods known in the art.See, e.g., WO 2009/067800, WO 2011/133886, and U.S. Patent ApplicationPublication No. 2014322129, incorporated by reference herein in theirentirety.

The variant polypeptides or immunomodulatory proteins of an IgSFconjugate may be “attached to” the effector moiety by any means by whichthe variant polypeptides or immunomodulatory proteins can be associatedwith, or linked to, the effector moiety. For example, the variantpolypeptides or immunomodulatory proteins of an IgSF conjugate may beattached to the effector moiety by chemical or recombinant means.Chemical means for preparing fusions or conjugates are known in the artand can be used to prepare the IgSF conjugate. The method used toconjugate the variant polypeptides or immunomodulatory proteins andeffector moiety must be capable of joining the variant polypeptides orimmunomodulatory proteins with the effector moiety without interferingwith the ability of the variant polypeptides or immunomodulatoryproteins to bind to their one or more counter structure ligands.

The variant polypeptides or immunomodulatory proteins of an IgSFconjugate may be linked indirectly to the effector moiety. For example,the variant polypeptides or immunomodulatory proteins of an IgSFconjugate may be directly linked to a liposome containing the effectormoiety of one of several types. The effector moiety(s) and/or thevariant polypeptides or immunomodulatory proteins may also be bound to asolid surface.

In some embodiments, the variant polypeptides or immunomodulatoryproteins of an IgSF conjugate and the effector moiety are both proteinsand can be conjugated using techniques well known in the art. There areseveral hundred crosslinkers available that can conjugate two proteins.(See for example “Chemistry of Protein Conjugation and Crosslinking,”1991, Shans Wong, CRC Press, Ann Arbor). The crosslinker is generallychosen based on the reactive functional groups available or inserted onthe variant polypeptides or immunomodulatory proteins and/or effectormoiety. In addition, if there are no reactive groups, a photoactivatiblecrosslinker can be used. In certain instances, it may be desirable toinclude a spacer between the variant polypeptides or immunomodulatoryproteins and the effector moiety. Crosslinking agents known to the artinclude the homobifunctional agents: glutaraldehyde, dimethyladipimidateand Bis(diazobenzidine) and the heterobifunctional agents: mMaleimidobenzoyl-N-Hydroxysuccinimide and Sulfo-mMaleimidobenzoyl-N-Hydroxysuccinimide.

In some embodiments, the variant polypeptides or immunomodulatoryproteins of an IgSF conjugate may be engineered with specific residuesfor chemical attachment of the effector moiety. Specific residues usedfor chemical attachment of molecule known to the art include lysine andcysteine. The crosslinker is chosen based on the reactive functionalgroups inserted on the variant polypeptides or immunomodulatoryproteins, and available on the effector moiety.

An IgSF conjugate may also be prepared using recombinant DNA techniques.In such a case a DNA sequence encoding the variant polypeptides orimmunomodulatory proteins is fused to a DNA sequence encoding theeffector moiety, resulting in a chimeric DNA molecule. The chimeric DNAsequence is transfected into a host cell that expresses the fusionprotein. The fusion protein can be recovered from the cell culture andpurified using techniques known in the art.

Examples of attaching an effector moiety, which is a label, to thevariant polypeptides or immunomodulatory proteins include the methodsdescribed in Hunter, et al., Nature 144:945 (1962); David, et al.,Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219(1981); Nygren, J. Histochem. and Cytochem. 30:407 (1982); Wensel andMeares, Radioimmunoimaging and Radioimmunotherapy, Elsevier, N.Y.(1983); and Colcher et al., “Use Of Monoclonal Antibodies asRadiopharmaceuticals For The Localization Of Human Carcinoma XenograftsIn Athymic Mice”, Meth. Enzymol., 121:802-16 (1986).

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as 99Tc or 1231, 186Re, 188Re and 111In can be attached viaa cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al., Biochem. Biophys.Res. Commun. 80:49-57 (1978)) can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the variant polypeptides or immunomodulatory proteins anda cytotoxic agent may be made using a variety of bifunctional proteincoupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate(SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate(SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters(such as dimethyl adipimidate HCl), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azidocompounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as toluene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Forexample, a ricin immunotoxin can be prepared as described in Vitetta etal., Science 238:1098 (1987). Carbon-14-labeled1-p-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid(MX-DTPA) is an exemplary chelating agent for conjugation ofradionucleotide to the antibody. See, e.g., WO94/11026. The linker maybe a “cleavable linker” facilitating release of the cytotoxic drug inthe cell. For example, an acid-labile linker, peptidase-sensitivelinker, photolabile linker, dimethyl linker or disulfide-containinglinker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No.5,208,020) may be used.

The IgSF conjugates of the invention expressly contemplate, but are notlimited to, drug conjugates prepared with cross-linker reagents: BMPS,EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH,sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC,and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) whichare commercially available (e.g., from Pierce Biotechnology, Inc.,Rockford, Ill., U.S.A). See pages 467-498, 2003-2004 ApplicationsHandbook and Catalog.

C. Transmembrane and Secretable Immunomodulatory Proteins and EngineeredCells

Provided herein are engineered cells which express the immunomodulatoryvariant ICOSL polypeptides (alternatively, “engineered cells”). In someembodiments, the variant ICOSL polypeptide is expressed on a cell, suchas an immune cell (e.g. T cell or antigen presenting cell), inmembrane-bound form, thereby providing a transmembrane immunomodulatoryprotein (hereinafter also called a “TIP”). In some aspects, the variantICOSL polypeptide is expressed in a cell, such as an immune cell (e.g. Tcell or antigen presenting cell), in secretable form to thereby producea secreted or soluble form of the variant ICOSL polypeptide (hereinafteralso called a “SIP”), such as when the cells are administered to asubject. In some aspects, a SIP can antagonize a cognate binding partnerin the environment (e.g. tumor microenvironment) in which it issecreted.

1. Transmembrane Immunomodulatory Proteins

In some embodiments, an immunomodulatory polypeptide comprising avariant ICOSL can be a membrane bound protein. As described in moredetail below, the immunomodulatory polypeptide can be a transmembraneimmunomodulatory polypeptide comprising a variant ICOSL in which iscontained: an ectodomain containing at least one affinity modified IgSFdomain (IgV or IgC), a transmembrane domain and, optionally, acytoplasmic domain. In some embodiments, the transmembraneimmunomodulatory protein can be expressed on the surface of an immunecell, such as a mammalian cell, including on the surface of a lymphocyte(e.g. T cell or NK cell) or antigen presenting cell. In someembodiments, the transmembrane immunomodulatory protein is expressed onthe surface of a mammalian T-cell, including such T-cells as: a T helpercell, a cytotoxic T-cell (alternatively, cytotoxic T lymphocyte or CTL),a natural killer T-cell, a regulatory T-cell, a memory T-cell, or agamma delta T-cell. In some embodiments, the mammalian cell is anantigen presenting cell (APC). Typically, but not exclusively, theectodomain (alternatively, “extracellular domain”) of comprises the oneor more amino acid variations (e.g. amino acid substitutions) of thevariant ICOSL of the invention. Thus, for example, in some embodiments atransmembrane protein will comprise an ectodomain that comprises one ormore amino acid substitutions of a variant ICOSL of the invention.

In some embodiments, the engineered cells express variant ICOSLpolypeptides that are transmembrane immunomodulatory polypeptides (TIPs)that can be a membrane protein such as a transmembrane protein. Intypical embodiments, the ectodomain of a membrane protein comprises anextracellular domain or IgSF domain thereof of a variant ICOSL providedherein in which is contained one or more amino acid substitutions in atleast one IgSF domain as described. The transmembrane immunomodulatoryproteins provided herein further contain a transmembrane domain linkedto the ectodomain. In some embodiments, the transmembrane domain resultsin an encoded protein for cell surface expression on a cell. In someembodiments, the transmembrane domain is linked directly to theectodomain. In some embodiments, the transmembrane domain is linkedindirectly to the ectodomain via one or more linkers or spacers. In someembodiments, the transmembrane domain contains predominantly hydrophobicamino acid residues, such as leucine and valine.

In some embodiments, a full length transmembrane anchor domain can beused to ensure that the TIPs will be expressed on the surface of theengineered cell, such as engineered T cell. Conveniently, this could befrom a particular native protein that is being affinity modified (e.g.ICOSL or other native IgSF protein), and simply fused to the sequence ofthe first membrane proximal domain in a similar fashion as the nativeIgSF protein (e.g. ICOSL). In some embodiments, the transmembraneimmunomodulatory protein comprises a transmembrane domain of thecorresponding reference (e.g., unmodified) or wild-type IgSF member,such as a transmembrane domain contained in the sequence of amino acidsset forth in SEQ ID NO:5 (Table 2). In some embodiments, the membranebound form comprises a transmembrane domain of the correspondingreference (e.g., unmodified) or wild-type polypeptide, such ascorresponding to residues 257-277 of SEQ ID NO:5.

In some embodiments, the transmembrane domain is a non-nativetransmembrane domain that is not the transmembrane domain of nativeICOSL. In some embodiments, the transmembrane domain is derived from atransmembrane domain from another non-ICOSL family member polypeptidethat is a membrane-bound or is a transmembrane protein. In someembodiments, a transmembrane anchor domain from another protein on Tcells can be used. In some embodiments, the transmembrane domain isderived from CD8. In some embodiments, the transmembrane domain canfurther contain an extracellular portion of CD8 that serves as a spacerdomain. An exemplary CD8 derived transmembrane domain is set forth inSEQ ID NO: 246 or 483 or a portion thereof containing the CD8transmembrane domain. In some embodiments, the transmembrane domain is asynthetic transmembrane domain.

In some embodiments, the transmembrane immunomodulatory protein furthercontains an endodomain, such as a cytoplasmic signaling domain, linkedto the transmembrane domain. In some embodiments, the cytoplasmicsignaling domain induces cell signaling. In some embodiments, theendodomain of the transmembrane immunomodulatory protein comprises thecytoplasmic domain of the corresponding reference (e.g., unmodified) orwild-type polypeptide, such as a cytoplasmic domain contained in thesequence of amino acids set forth in SEQ ID NO:5 (see Table 2).

In some embodiments, a provided transmembrane immunomodulatory proteinthat is or comprises a variant ICOSL comprises a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 257 andcontains an ectodomain comprising at least one affinity-modified ICOSLIgSF domain as described and a transmembrane domain. In someembodiments, the transmembrane immunomodulatory protein contains any oneor more amino acid substitutions in an IgSF domain (e.g. IgV domain) asdescribed, including any set forth in Table 1. In some embodiments, thetransmembrane immunomodulatory protein can further comprise acytoplasmic domain as described. In some embodiments, the transmembraneimmunomodulatory protein can further contain a signal peptide. In someembodiments, the signal peptide is the native signal peptide ofwild-type IgSF member, such as contained in the sequence of amino acidsset forth in SEQ ID NO: 5 (see e.g. Table 2).

In some embodiments, provided are transmembrane immunomodulatoryproteins comprising the amino acid substitutionsE16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R, N52H/N57Y/Q100R, orN52H/N57Y/Q100P. In some embodiments, the provided transmembraneimmunomodulatory protein is or comprises a variant ICOSL comprising thesequence of amino acids set forth in SEQ ID NO:257, but in which iscontained amino substitutionsE16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R, N52H/N57Y/Q100R, orN52H/N57Y/Q100P at corresponding positions in SEQ ID NO:257, or asequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity toSEQ ID NO: 257 and contains the amino acid substitutionsE16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R, N52H/N57Y/Q100R, orN52H/N57Y/Q100P.

In some embodiments, provided are transmembrane immunomodulatoryproteins comprising the sequence of amino acids set forth in SEQ ID NOS:496 or 497 (each containing the amino acid substitution N52D), SEQ IDNOS: 498 or 499 (each containing the amino acid substitutionsN52H/N57Y/Q100P), SEQ ID NOS: 500 or 501 (each containing the amino acidsubstitutions E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R) or SEQID NOS: 502 or 503 (each containing the amino acid substitutionsN52H/N57Y/Q100R), or a sequence of amino acids that comprises at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity to any of SEQ ID NOS: 495-503 and that containsthe indicated amino acid substitutions. In some embodiments, whenexpressed in an engineered cell, such transmembrane immunomodulatoryproteins are expressed on the surface of the cell.

Also provided is a nucleic acid molecule encoding such transmembraneimmunomodulatory proteins. In some embodiments, a nucleic acid moleculeencoding a transmembrane immunomodulatory protein comprises a nucleotidesequence that encodes a sequence of amino acids that exhibits at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity to SEQ ID NO: 257 and contains an ectodomaincomprising at least one affinity-modified IgSF domain as described, atransmembrane domain and, optionally, a cytoplasmic domain. In someembodiments, the nucleic acid molecule can further comprise a sequenceof nucleotides encoding a signal peptide. In some embodiments, thesignal peptide is the native signal peptide of the correspondingwild-type IgSF member (see e.g. Table 2).

Exemplary of a transmembrane immunomodulatory protein is a ICOSL TIPcomprising i) the sequence of amino acids set forth in SEQ ID NO:383 orii) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to SEQ ID NO: 243 and that comprises the affinity-modifieddomain contained in SEQ ID NO: 243 or the amino acid substitutionstherein. Also provided is i) a sequence of nucleotides set forth in SEQID NO: 244, ii) a sequence that exhibits at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to SEQ ID NO: 244 and that encodes a TIP that comprises theaffinity-modified domain of SEQ ID NO: 243, or iii) a sequence of i) orii) having degenerate codons.

In some embodiments, provided are CAR-related transmembraneimmunomodulatory proteins in which the endodomain of a transmembraneimmunomodulatory protein comprises a cytoplasmic signaling domain thatcomprises at least one ITAM (immunoreceptor tyrosine-based activationmotif)-containing signaling domain. ITAM is a conserved motif found in anumber of protein signaling domains involved in signal transduction ofimmune cells, including in the CD3-zeta chain (“CD3-z”) involved inT-cell receptor signal transduction. In some embodiments, the endodomaincomprises at CD3-zeta signaling domain. In some embodiments, theCD3-zeta signaling domain comprises the sequence of amino acids setforth in SEQ ID NO: 243 or a sequence of amino acids that exhibits atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% to SEQ ID NO:247 and retains the activity of T cellsignaling. In some embodiments, the endodomain of a CAR-relatedtransmembrane immunomodulatory protein can further comprise acostimulatory signaling domain to further modulate immunomodulatoryresponses of the T-cell. In some embodiments, the costimulatorysignaling domain is CD28, ICOS, 41BB or OX40. In some embodiments, thecostimulatory signaling domain is a derived from CD28 or 4-1BB andcomprises the sequence of amino acids set forth in any of SEQ ID NOS:484-487 or a sequence of amino acids that exhibits at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to SEQID NO: 484-487 and retains the activity of T cell costimulatorysignaling. In some embodiments, the provided CAR-related transmembraneimmunomodulatory proteins have features of CARs to stimulate T cellsignaling upon binding of an affinity modified IgSF domain to a cognatebinding partner or counter structure. In some embodiments, upon specificbinding by the affinity-modified IgSF domain to its counter structurecan lead to changes in the immunological activity of the T-cell activityas reflected by changes in cytotoxicity, proliferation or cytokineproduction.

In some embodiments, the transmembrane immunomodulatory protein does notcontain an endodomain capable of mediating cytoplasmic signaling. Insome embodiments, the transmembrane immunomodulatory protein lacks thesignal transduction mechanism of the wild-type or unmodified polypeptideand therefore does not itself induce cell signaling. In someembodiments, the transmembrane immunomodulatory protein lacks anintracellular (cytoplasmic) domain or a portion of the intracellulardomain of the corresponding reference (e.g., unmodified) or wild-typepolypeptide, such as a cytoplasmic signaling domain contained in thesequence of amino acids set forth in SEQ ID NO:5 (see Table 2). In someembodiments, the transmembrane immunomodulatory protein does not containan ITIM (immunoreceptor tyrosine-based inhibition motif), such ascontained in certain inhibitory receptors, including inhibitoryreceptors of the IgSF family (e.g. PD-1 or TIGIT). Thus, in someembodiments, the transmembrane immunomodulatory protein only containsthe ectodomain and the transmembrane domain, such as any as described.

2. Secreted Immunomodulatory Proteins and Engineered Cells

In some embodiments, the ICOSL variant immunomodulatory polypeptidecontaining any one or more of the amino acid mutations as describedherein, is secretable, such as when expressed from a cell. Such avariant ICOSL immunomodulatory protein does not comprise a transmembranedomain. In some embodiments, the variant ICOSL immunomodulatory proteinis not conjugated to a half-life extending moiety (such as an Fc domainor a multimerization domain). In some embodiments, the variant ICOSLimmunomodulatory protein comprises a signal peptide, e.g. an antibodysignal peptide or other efficient signal sequence to get domains outsideof cell. When the immunomodulatory protein comprises a signal peptideand is expressed by an engineered cell, the signal peptide causes theimmunomodulatory protein to be secreted by the engineered cell.Generally, the signal peptide, or a portion of the signal peptide, iscleaved from the immunomodulatory protein with secretion. Theimmunomodulatory protein can be encoded by a nucleic acid (which can bepart of an expression vector). In some embodiments, the immunomodulatoryprotein is expressed and secreted by a cell (such as an immune cell, forexample a primary immune cell).

Thus, in some embodiments, there are provided variant ICOSLimmunomodulatory proteins that further comprise a signal peptide. Insome embodiments, provided herein is a nucleic acid molecule encodingthe variant ICOSL immunomodulatory protein operably connected to asecretion sequence encoding the signal peptide.

A signal peptide is a sequence on the N-terminus of an immunomodulatoryprotein that signals secretion of the immunomodulatory protein from acell. In some embodiments, the signal peptide is about 5 to about 40amino acids in length (such as about 5 to about 7, about 7 to about 10,about 10 to about 15, about 15 to about 20, about 20 to about 25, orabout 25 to about 30, about 30 to about 35, or about 35 to about 40amino acids in length).

In some embodiments, the signal peptide is a native signal peptide fromthe corresponding wild-type ICOSL (see Table 6). In some embodiments,the signal peptide is a non-native signal peptide. For example, in someembodiments, the non-native signal peptide is a mutant native signalpeptide from the corresponding wild-type ICOSL, and can include one ormore (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) substitutionsinsertions or deletions. In some embodiments, the non-native signalpeptide is a signal peptide or mutant thereof of a family member fromthe same IgSF family as the wild-type IgSF family member. In someembodiments, the non-native signal peptide is a signal peptide or mutantthereof from an IgSF family member from a different IgSF family than thewild-type IgSF family member. In some embodiments, the signal peptide isa signal peptide or mutant thereof from a non-IgSF protein family, suchas a signal peptide from an immunoglobulin (such as IgG heavy chain orIgG-kappa light chain), a cytokine (such as interleukin-2 (IL-2), orCD33), a serum albumin protein (e.g. HSA or albumin), a human azurocidinpreprotein signal sequence, a luciferase, a trypsinogen (e.g.chymotrypsinogen or trypsinogen) or other signal peptide able toefficiently secrete a protein from a cell. Exemplary signal peptidesinclude any described in the Table 6.

TABLE 6 Exemplary Signal Peptides SEQ ID NO Signal PeptidePeptide Sequence SEQ ID NO: 346 HSA signal peptide MKWVTFISLLFLFSSAYSSEQ ID NO: 347 Ig kappa light chain MDMRAPAGIFGFLLVLFPGYRSSEQ ID NO: 348 human azurocidin preprotein MTRLTVLALLAGLLASSRAsignal sequence SEQ ID NO: 349 IgG heavy chain signal peptideMELGLSWIFLLAILKGVQC SEQ ID NO: 350 IgG heavy chain signal peptideMELGLRWVFLVAILEGVQC SEQ ID NO: 351 IgG heavy chain signal peptideMKHLWFFLLLVAAPRWVLS SEQ ID NO: 352 IgG heavy chain signal peptideMDWTWRILFLVAAATGAHS SEQ ID NO: 353 IgG heavy chain signal peptideMDWTWRFLFVVAAATGVQS SEQ ID NO: 354 IgG heavy chain signal peptideMEFGLSWLFLVAILKGVQC SEQ ID NO: 355 IgG heavy chain signal peptideMEFGLSWVFLVALFRGVQC SEQ ID NO: 356 IgG heavy chain signal peptideMDLLHKNMKHLWFFLLLVAAPRWV LS SEQ ID NO: 357 IgG Kappa light chain signalMDMRVPAQLLGLLLLWLSGARC sequence SEQ ID NO: 358IgG Kappa light chain signal MKYLLPTAAAGLLLLAAQPAMA sequenceSEQ ID NO: 359 Gaussia luciferase MGVKVLFALICIAVAEA SEQ ID NO: 360Human albumin MKWVTFISLLFLFSSAYS SEQ ID NO: 361 Human chymotrypsinogenMAFLWLLSCWALLGTTFG SEQ ID NO: 362 Human interleukin-2 MQLLSCIALILALVSEQ ID NO: 363 Human trypsinogen-2 MNLLLILTFVAAAVA

In some embodiments of a secretable variant ICOSL immunomodulatoryprotein, the immunomodulatory protein comprises a signal peptide whenexpressed, and the signal peptide (or a portion thereof) is cleaved fromthe immunomodulatory protein upon secretion.

In some embodiments, the engineered cells express a variant ICOSLpolypeptide that is secreted from the cell. In some embodiments, such avariant ICOSL polypeptide is encoded by a nucleic acid molecule encodingan immunomodulatory protein under the operable control of a signalsequence for secretion. In some embodiments, the encodedimmunomodulatory protein is secreted when expressed from a cell. In someembodiments, the immunomodulatory protein encoded by the nucleic acidmolecule does not comprise a transmembrane domain. In some embodiments,the immunomodulatory protein encoded by the nucleic acid molecule doesnot comprise a half-life extending moiety (such as an Fc domain or amultimerization domain). In some embodiments, the immunomodulatoryprotein encoded by the nucleic acid molecule comprises a signal peptide.In some embodiments, a nucleic acid of the invention further comprisesnucleotide sequence that encodes a secretory or signal peptide operablylinked to the nucleic acid encoding the immunomodulatory protein,thereby allowing for secretion of the immunomodulatory protein

3. Cells and Engineering Cells

Provided herein are engineered cells expressing any of the providedimmunomodulatory polypeptides. In some embodiments, the engineered cellsexpress on their surface any of the provided transmembraneimmunomodulatory polypeptides. In some embodiments, the engineered cellsexpress and are capable of or are able to secrete the immunomodulatoryprotein from the cells under conditions suitable for secretion of theprotein. In some embodiments, the immunomodulatory protein is expressedon or in a lymphocyte such as a tumor infiltrating lymphocyte (TIL),T-cell or NK cell, or on a myeloid cell. In some embodiments, theengineered cells are antigen presenting cells (APCs). In someembodiments, the engineered cells are engineered mammalian T-cells orengineered mammalian antigen presenting cells (APCs). In someembodiments, the engineered T-cells or APCs are human or murine cells.

In some embodiments, engineered T-cells include, but are not limited to,T helper cell, cytotoxic T-cell (alternatively, cytotoxic T lymphocyteor CTL), natural killer T-cell, regulatory T-cell, memory T-cell, orgamma delta T-cell. In some embodiments, the engineered T cells are CD4+or CD8+. In addition to the signal of the MHC, engineered T-cells alsorequire a co-stimulatory signal which in some embodiments is provided bya variant ICOSL transmembrane immunomodulatory polypeptide expressed inmembrane bound form as discussed previously.

In some embodiments, the engineered APCs include, for example, MHC IIexpressing APCs such as macrophages, B cells, and dendritic cells, aswell as artificial APCs (aAPCs) including both cellular and acellular(e.g., biodegradable polymeric microparticles) aAPCs. Artificial APCs(aAPCs) are synthetic versions of APCs that can act in a similar mannerto APCs in that they present antigens to T-cells as well as activatethem. Antigen presentation is performed by the MHC (Class I or ClassII). In some embodiments, in engineered APCs such as aAPCs, the antigenthat is loaded onto the MHC is, in some embodiments, a tumor specificantigen or a tumor associated antigen. The antigen loaded onto the MHCis recognized by a T-cell receptor (TCR) of a T cell, which, in somecases, can express ICOS, CD28, or other molecule recognized by thevariant ICOSL polypeptides provided herein. Materials which can be usedto engineer an aAPC include: poly (glycolic acid),poly(lactic-co-glycolic acid), iron-oxide, liposomes, lipid bilayers,sepharose, and polystyrene.

In some embodiments a cellular aAPC can be engineered to contain a TIPand TCR agonist which is used in adoptive cellular therapy. In someembodiments, a cellular aAPC can be engineered to contain a TIP and TCRagonist which is used in ex vivo expansion of human T cells, such asprior to administration, e.g., for reintroduction into the patient. Insome aspects, the aAPC may include expression of at least one anti-CD3antibody clone, e.g. such as, for example, OKT3 and/or UCHT1. In someaspects, the aAPCs may be inactivated (e.g. irradiated). In someembodiment, the TIP can include any variant IgSF domain that exhibitsbinding affinity for a cognate binding partner on a T cell.

In some embodiments, an immunomodulatory protein provided herein, suchas a transmembrane immunomodulatory protein or a secretableimmunomodulatory protein, is co-expressed or engineered into a cell thatexpresses an antigen-binding receptor, such as a recombinant receptor,such as a chimeric antigen receptor (CAR) or T cell receptor (TCR). Insome embodiments, the engineered cell, such as an engineered T cell,recognizes a desired antigen associated with cancer, inflammatory andautoimmune disorders, or a viral infection. In specific embodiments, theantigen-binding receptor contains an antigen-binding moiety thatspecifically binds a tumor specific antigen or a tumor associatedantigen. In some embodiments, the engineered T-cell is a CAR (chimericantigen receptor) T-cell that contains an antigen-binding domain (e.g.scFv) that specifically binds to an antigen, such as a tumor specificantigen or tumor associated antigen. In some embodiments, theantigen-binding domain (e.g. scFv) is specific for a particular antigen,e.g., CD19. Exemplary of a CAR is an anti-CD19 CAR, such as a CARcontaining an anti-CD19 scFv set forth in SEQ ID NO:482 or SEQ IDNO:245. In some embodiments, the TIP protein is expressed in anengineered T-cell receptor cell or an engineered chimeric antigenreceptor cell. In such embodiments, the engineered cell co-expresses theTIP and the CAR or TCR. In some embodiments, the SIP protein isexpressed in an engineered T-cell receptor cell or an engineeredchimeric antigen receptor cell. In such embodiments, the engineered cellco-expresses the SIP and the CAR or TCR.

Chimeric antigen receptors (CARs) are recombinant receptors that includean antigen-binding domain (ectodomain), a transmembrane domain and anintracellular signaling region (endodomain) that is capable of inducingor mediating an activation signal to the T cell after the antigen isbound. In some example, CAR-expressing cells are engineered to expressan extracellular single chain variable fragment (scFv) with specificityfor a particular tumor antigen linked to an intracellular signaling partcomprising an activating domain and, in some cases, a costimulatorydomain. The costimulatory domain can be derived from, e.g., CD28, OX-40,4-1BB/CD137 or inducible T cell costimulator (ICOS). The activatingdomain can be derived from, e.g., CD3, such as CD3 zeta, epsilon, delta,gamma, or the like. In certain embodiments, the CAR is designed to havetwo, three, four, or more costimulatory domains. The CAR scFv can bedesigned to target an antigen expressed on a cell associated with adisease or condition, e.g. a tumor antigen, such as, for example, CD19,which is a transmembrane protein expressed by cells in the B celllineage, including all normal B cells and B cell malignances, includingbut not limited to NHL, CLL, and non-T cell ALL. Example CAR+ T celltherapies and constructs are described in U.S. Patent Publication Nos.2013/0287748, 2014/0227237, 2014/0099309, and 2014/0050708, and thesereferences are incorporated by reference in their entirety.

In some aspects, the antigen-binding domain is an antibody orantigen-binding fragment thereof, such as a single chain fragment(scFv). In some embodiments, the antigen is expressed on a tumor orcancer cell. Exemplary of an antigen is CD19. Exemplary of a CAR is ananti-CD19 CAR, such as a CAR containing an anti-CD19 scFv set forth inSEQ ID NO: 245. In some embodiments, the CAR further contains a spacer,a transmembrane domain, and an intracellular signaling domain or regioncomprising an ITAM signaling domain, such as a CD3zeta signaling domain.In some embodiments, the CAR further includes a costimulatory signalingdomain.

In some embodiments, the CAR further contains a spacer or hinge, atransmembrane domain, and an intracellular signaling domain (endodomain)comprising an ITAM signaling domain, such as a CD3zeta signaling domain.In some embodiments, the CAR further includes a costimulatory signalingdomain. The costimulatory domain can be derived from, e.g., CD28, OX-40,4-1BB/CD137 or inducible T cell costimulator (ICOS). In certainembodiments, the CAR is designed to have two, three, four, or morecostimulatory domains. The CAR scFv can be designed to target an antigenexpressed on a cell associated with a disease or condition, e.g. a tumorantigen, such as, for example, CD19, which is a transmembrane proteinexpressed by cells in the B cell lineage, including all normal B cellsand B cell malignances, including but not limited to NHL, CLL, and non-Tcell ALL. Example CAR+ T cell therapies and constructs are described inU.S. Patent Publication Nos. 2013/0287748, 2014/0227237, 2014/0099309,and 2014/0050708, and these references are incorporated by reference intheir entirety. In some embodiments, the spacer or hinge is presentbetween the antigen-binding domain and the transmembrane domain, such asis between the antigen-binding domain and plasma membrane when expressedon a cell. In some embodiments, the spacer or hinge is derived from IgGsubclass (such as IgG1 and IgG4, IgD or CD8 (see e.g., Qin et al. (2017)J. Hematol. Oncol., 10:68). In some embodiments, the spacer or hinge isderived from IgG1.

In some embodiments, the spacer and transmembrane domain are the hingeand transmembrane domain derived from CD8, such as having an exemplarysequence set forth in SEQ ID NO: 246, 483, or 897 or a sequence of aminoacids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ IDNO:246, 483, or 897. In some embodiments, the endodomain comprises atCD3-zeta signaling domain. In some embodiments, the CD3-zeta signalingdomain comprises the sequence of amino acids set forth in SEQ ID NO: 243or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequenceidentity to SEQ ID NO: 247 and retains the activity of T cell signaling.In some embodiments, the endodomain of a CAR can further comprise acostimulatory signaling domain or region to further modulateimmunomodulatory responses of the T-cell. In some embodiments, thecostimulatory signaling domain is or comprises a costimulatory region,or is derived from a costimulatory region, of CD28, ICOS, 41BB or OX40.In some embodiments, the costimulatory signaling domain is a derivedfrom CD28 or 4-1BB and comprises the sequence of amino acids set forthin any of SEQ ID NOS: 484-487 or a sequence of amino acids that exhibitsat least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% or more sequence identity to SEQ ID NO:484-487 andretains the activity of T cell costimulatory signaling.

Provided herein is a polynucleotide encoding an ICOSL polypeptide andencoding one or more proteins, such as a recombinant antigen receptor(e.g., chimeric antigen receptor (CAR) or engineered T cell receptor(TCR)), a marker, and one or more self-cleaving peptides. In someembodiments, the construct encoding the CAR further encodes a secondprotein, such as a marker, e.g. detectable protein, separated from theCAR by a self-cleaving peptide sequence. In some examples, the nucleicacid encoding the variant ICOSL polypeptide is separated from the one ormore sequence(s) that is a nucleic acid encoding a protein, wherein theprotein encodes a recombinant antigen receptor (e.g., CAR or TCR), amarker, a cytokine, or a chemokine. Any of the nucleotide sequences canbe in a vector, such as viral vector. In some examples, the viral vectoris a lentiviral vector or retroviral vector.

In some embodiments, the self-cleaving peptide sequence is an F2A, T2A,E2A or P2A self-cleaving peptide. Exemplary sequences of a T2Aself-cleaving peptide are set for the in any one of SEQ ID NOS: 250,488, 860-862 or a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%or more sequence identity to any of SEQ ID NOS: 250, 488, 860-862. Insome embodiments, the T2A is encoded by the sequence of nucleotides setforth in SEQ ID NO:249 or a sequence that exhibits at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% ormore sequence identity to any of SEQ ID NO: 249. An exemplary sequenceof a P2A self-cleaving peptide is set in SEQ ID NO: 863 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity toSEQ ID NOS: 863. In some cases, a nucleic acid construct that encodesmore than one P2A self-cleaving peptide (such as a P2A1 and P2A2), inwhich the nucleotide sequence P2A1 and P2A2 each encode the P2A setforth in SEQ ID NO:863, the nucleotide sequence may be different toavoid recombination between sequences.

In some embodiments, the marker is a detectable protein, such as afluorescent protein, e.g., a green fluorescent protein (GFP) or bluefluorescent protein (BFP). Exemplary sequences of a fluorescent proteinmarker are set forth in SEQ ID NO:489, 858, 859, 903 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity toSEQ ID NO: 489, 858, 859, 903.

In some embodiments, the CAR is an anti-CD19 CAR that has the sequenceof amino acids set forth in any of SEQ ID NOS: 479, 490, 491, 492, 898,899, 901, or 902 or a sequence of amino acids that exhibits at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% or more sequence identity to any one of SEQ ID NOS: 479, 490, 491,492, 898, 899, 901, or 902. In some embodiments, the CAR is encoded by asequence of nucleotides set forth in SEQ ID NO: 248 or 900 or a sequenceof amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity toany one of SEQ ID NO: 248 or 900.

In another embodiment, the engineered T-cell possesses a TCR, includinga recombinant or engineered TCR. In some embodiments, the TCR can be anative TCR. Those of skill in the art will recognize that generallynative mammalian T-cell receptors comprise an alpha and a beta chain (ora gamma and a delta chain) involved in antigen specific recognition andbinding. In some embodiments, the TCR is an engineered TCR that ismodified. In some embodiments, the TCR of an engineered T-cellspecifically binds to a tumor associated or tumor specific antigenpresented by an APC.

In some embodiments, the immunomodulatory polypeptides, such astransmembrane immunomodulatory polypeptides or secretableimmunomodulatory polypeptides, can be incorporated into engineeredcells, such as engineered T cells or engineered APCs, by a variety ofstrategies such as those employed for recombinant host cells. A varietyof methods to introduce a DNA construct into primary T cells are knownin the art. In some embodiments, viral transduction or plasmidelectroporation are employed. In typical embodiments, the nucleic acidmolecule encoding the immunomodulatory protein, or the expressionvector, comprises a signal peptide that localizes the expressedtransmembrane immunomodulatory proteins to the cellular membrane or forsecretion. In some embodiments, a nucleic acid encoding a transmembraneimmunomodulatory proteins of the invention is sub-cloned into a viralvector, such as a retroviral vector, which allows expression in the hostmammalian cell. The expression vector can be introduced into a mammalianhost cell and, under host cell culture conditions, the immunomodulatoryprotein is expressed on the surface or is secreted.

In an exemplary example, primary T-cells can be purified ex vivo (CD4cells or CD8 cells or both) and stimulated with an activation protocolconsisting of various TCR/CD28 agonists, such as anti-CD3/anti-CD28coated beads. After a 2 or 3 day activation process, a recombinantexpression vector containing an immunomodulatory polypeptide can bestably introduced into the primary T cells through art standardlentiviral or retroviral transduction protocols or plasmidelectroporation strategies. Cells can be monitored for immunomodulatorypolypeptide expression by, for example, flow cytometry usinganti-epitope tag or antibodies that cross-react with native parentalmolecule and polypeptides comprising variant ICOSL. T-cells that expressthe immunomodulatory polypeptide can be enriched through sorting withanti-epitope tag antibodies or enriched for high or low expressiondepending on the application.

Upon immunomodulatory polypeptide expression the engineered T-cell canbe assayed for appropriate function by a variety of means. Theengineered CAR or TCR co-expression can be validated to show that thispart of the engineered T cell was not significantly impacted by theexpression of the immunomodulatory protein. Once validated, standard invitro cytotoxicity, proliferation, or cytokine assays (e.g., IFN-gammaexpression) can be used to assess the function of engineered T-cells.Exemplary standard endpoints are percent lysis of the tumor line,proliferation of the engineered T-cell, or IFN-gamma protein expressionin culture supernatants. An engineered construct which results instatistically significant increased lysis of tumor line, increasedproliferation of the engineered T-cell, or increased IFN-gammaexpression over the control construct can be selected for. Additionally,non-engineered, such as native primary or endogenous T-cells could alsobe incorporated into the same in vitro assay to measure the ability ofthe immunomodulatory polypeptide construct expressed on the engineeredcells, such as engineered T-cells, to modulate activity, including, insome cases, to activate and generate effector function in bystander,native T-cells. Increased expression of activation markers such as CD69,CD44, or CD62L could be monitored on endogenous T cells, and increasedproliferation and/or cytokine production could indicate desired activityof the immunomodulatory protein expressed on the engineered T cells.

In some embodiments, the similar assays can be used to compare thefunction of engineered T cells containing the CAR or TCR alone to thosecontaining the CAR or TCR and a TIP construct. Typically, these in vitroassays are performed by plating various ratios of the engineered T celland a “tumor” cell line containing the cognate CAR or TCR antigentogether in culture. Standard endpoints are percent lysis of the tumorline, proliferation of the engineered T cell, or IFN-gamma production inculture supernatants. An engineered immunomodulatory protein whichresulted in statistically significant increased lysis of tumor line,increased proliferation of the engineered T cell, or increased IFN-gammaproduction over the same TCR or CAR construct alone can be selected for.

Engineered human T cells can be analyzed in immunocompromised mice, likethe NSG strain, which lacks mouse T, NK and B cells. Engineered human Tcells in which the CAR or TCR binds a target counter-structure on thexenograft and is co-expressed with the TIP affinity modified IgSF domaincan be adoptively transferred in vivo at different cell numbers andratios compared to the xenograft. For example, engraftment of CD19+leukemia tumor lines containing a luciferase/GFP vector can be monitoredthrough bioluminescence or ex vivo by flow cytometry. In a commonembodiment, the xenograft is introduced into the murine model, followedby the engineered T cells several days later. Engineered T cellscontaining the immunomodulatory protein can be assayed for increasedsurvival, tumor clearance, or expanded engineered T cells numbersrelative to engineered T cells containing the CAR or TCR alone. As inthe in vitro assay, endogenous, native (i.e., non-engineered) human Tcells could be co-adoptively transferred to look for successful epitopespreading in that population, resulting in better survival or tumorclearance.

D. Infectious Agents Expressing Variant Polypeptides andImmunomodulatory Proteins

Also provided are infectious agents that contain nucleic acids encodingany of the variant polypeptides, such as ICOSL vIgD polypeptides,including secretable or transmembrane immunomodulatory proteinsdescribed herein. In some embodiments, such infectious agents candeliver the nucleic acids encoding the variant immunomodulatorypolypeptides described herein, such as ICOSL vIgD polypeptides, to atarget cell in a subject, e.g., immune cell and/or antigen-presentingcell (APC) or tumor cell in a subject. Also provided are nucleic acidscontained in such infectious agents, and/or nucleic acids for generationor modification of such infectious agents, such as vectors and/orplasmids, and compositions containing such infectious agents. In someembodiments, a variant ICOSL polypeptide is expressed in an infectiousagent (e.g. viral or bacterial agent) which, upon administration to asubject, is able to infect a cell in vivo, such as an immune cell (e.g.T cell or antigen presenting cell) or tumor, for delivery or expressionof the variant polypeptide as a TIP or a SIP in the cell.

In some embodiments, the infectious agent is a microorganism or amicrobe. In some embodiments, the infectious agent is a virus or abacterium. In some embodiments, the infectious agent is a virus. In someembodiments, the infectious agent is a bacterium. In some embodiments,such infectious agents can deliver nucleic acid sequences encoding anyof the variant polypeptides, such as ICOSL vIgD polypeptides, includingsecretable or transmembrane immunomodulatory proteins, described herein.Thus, in some embodiments, the cell in a subject that is infected orcontacted by the infectious agents can be rendered to express on thecell surface or secrete, the variant immunomodulatory polypeptides. Insome embodiments, the infectious agent can also deliver one or moreother therapeutics or nucleic acids encoding other therapeutics to thecell and/or to an environment within the subject. In some embodiments,other therapeutics that can be delivered by the infectious agentsinclude cytokines or other immunomodulatory molecules.

In some embodiments, the infectious agent, e.g., virus or bacteria,contains nucleic acid sequences that encode any of the variantpolypeptides, such as ICOSL vIgD polypeptides, including secretable ortransmembrane immunomodulatory proteins, described herein, and by virtueof contact and/or infection of a cell in the subject, the cell expressesthe variant polypeptides, such as ICOSL vIgD polypeptides, includingsecretable or transmembrane immunomodulatory proteins, encoded by thenucleic acid sequences contained in the infectious agent. In someembodiments, the infectious agent can be administered to the subject. Insome embodiments, the infectious agent can be introduced to cells fromthe subject ex vivo.

In some embodiments, the variant polypeptides, such as ICOSL vIgDpolypeptides, including transmembrane immunomodulatory proteins,expressed by the cell infected by the infectious agent is atransmembrane protein and is surface expressed. In some embodiments, thevariant polypeptides, such as ICOSL vIgD polypeptides, includingsecretable immunomodulatory proteins, expressed by the cell infected bythe infectious agent is expressed and secreted from the cell. Thetransmembrane immunomodulatory protein or secreted immunomodulatoryprotein can be any described herein.

In some embodiments, the cells in the subject that are targeted by theinfectious agent include a tumor cell, an immune cell, and/or anantigen-presenting cell (APC). In some embodiments, the infectious agenttargets a cell in the tumor microenvironment (TME). In some embodiments,the infectious agent delivers the nucleic acids encoding the variantpolypeptides, such as ICOSL vIgD polypeptides, including secretable ortransmembrane immunomodulatory proteins, to an appropriate cell (forexample, an APC, such as a cell that displays a peptide/MHC complex onits cell surface, such as a dendritic cell) or tissue (e.g., lymphoidtissue) that modulate an immune response and/or a specificcell-medicated immune response, e.g., CD4 and/or CD8 T cell response,which CD8 T cell response may include a cytotoxic T cell (CTL) response.In some embodiments, the infectious agent targets an APC, such as adendritic cell (DC). In some embodiments, the nucleic acid moleculedelivered by the infectious agents described herein include appropriatenucleic acid sequences necessary for the expression of the operablylinked coding sequences encoding the variant immunomodulatorypolypeptides, in a particular target cell, e.g., regulatory elementssuch as promoters.

In some embodiments, the infectious agent that contains nucleic acidsequences encoding the immunomodulatory polypeptides can also containnucleic acid sequences that encode one or more additional gene products,e.g., cytokines, prodrug converting enzymes, cytotoxins and/ordetectable gene products. For example, in some embodiments, theinfectious agent is an oncolytic virus and the virus can include nucleicacid sequences encoding additional therapeutic gene products (see, e.g.,Kim et al., (2009) Nat Rev Cancer 9:64-71; Garcia-Aragoncillo et al.,(2010) Curr Opin Mol Ther 12:403-411; see U.S. Pat. Nos. 7,588,767,7,588,771, 7,662,398 and 7,754,221 and U.S. Pat. Publ. Nos.2007/0202572, 2007/0212727, 2010/0062016, 2009/0098529, 2009/0053244,2009/0155287, 2009/0117034, 2010/0233078, 2009/0162288, 2010/0196325,2009/0136917 and 2011/0064650. In some embodiments, the additional geneproduct can be a therapeutic gene product that can result in death ofthe target cell (e.g., tumor cell) or gene products that can augment orboost or regulate an immune response (e.g., cytokine). Exemplary geneproducts also include among an anticancer agent, an anti-metastaticagent, an antiangiogenic agent, an immunomodulatory molecule, an immunecheckpoint inhibitor, an antibody, a cytokine, a growth factor, anantigen, a cytotoxic gene product, a pro-apoptotic gene product, ananti-apoptotic gene product, a cell matrix degradative gene, genes fortissue regeneration and reprogramming human somatic cells topluripotency, and other genes described herein or known to one of skillin the art. In some embodiments, the additional gene product isGranulocyte-macrophage colony-stimulating factor (GM-CSF).

1. Viruses

In some embodiments, the infectious agent is a virus. In someembodiments, the infectious agent is an oncolytic virus, or a virus thattargets particular cells, e.g., immune cells. In some embodiments, theinfectious agent targets a tumor cell and/or cancer cell in the subject.In some embodiments, the infectious agent targets an immune cell or anantigen-presenting cell (APC).

In some embodiments, the infectious agent is an oncolytic virus.Oncolytic viruses are viruses that accumulate in tumor cells andreplicate in tumor cells. By virtue of replication in the tumor cells,and optional delivery of nucleic acids encoding variant ICOSLpolypeptides or immunomodulatory polypeptides described herein, tumorcells are lysed, and the tumor shrinks and can be eliminated. Oncolyticviruses can also have a broad host and cell type range. For example,oncolytic viruses can accumulate in immunoprivileged cells orimmunoprivileged tissues, including tumors and/or metastases, and alsoincluding wounded tissues and cells, thus allowing the delivery andexpression of nucleic acids encoding the variant immunomodulatorypolypeptides described herein in a broad range of cell types. Oncolyticviruses can also replicate in a tumor cell specific manner, resulting intumor cell lysis and efficient tumor regression.

Exemplary oncolytic viruses include adenoviruses, adeno-associatedviruses, herpes viruses, Herpes Simplex Virus, vesticular stomaticvirus, Reovirus, Newcastle Disease virus, parvovirus, measles virus,vesticular stomatitis virus (VSV), Coxsackie virus and Vaccinia virus.In some embodiments, oncolytic viruses can specifically colonize solidtumors, while not infecting other organs, and can be used as aninfectious agent to deliver the nucleic acids encoding the variantimmunomodulatory polypeptides described herein to such solid tumors.

Oncolytic viruses for use in delivering the nucleic acids encodingvariant ICOSL polypeptides or immunomodulatory polypeptides describedherein, can be any of those known to one of skill in the art andinclude, for example, vesicular stomatitis virus, see, e.g., U.S. Pat.Nos. 7,731,974, 7,153,510, 6,653,103 and U.S. Pat. Pub. Nos.2010/0178684, 2010/0172877, 2010/0113567, 2007/0098743, 20050260601,20050220818 and EP Pat. Nos. 1385466, 1606411 and 1520175; herpessimplex virus, see, e.g., U.S. Pat. Nos. 7,897,146, 7,731,952,7,550,296, 7,537,924, 6,723,316, 6,428,968 and U.S. Pat. Pub. Nos.,2014/0154216, 2011/0177032, 2011/0158948, 2010/0092515, 2009/0274728,2009/0285860, 2009/0215147, 2009/0010889, 2007/0110720, 2006/0039894,2004/0009604, 2004/0063094, International Patent Pub. Nos., WO2007/052029, WO 1999/038955; retroviruses, see, e.g., U.S. Pat. Nos.6,689,871, 6,635,472, 5,851,529, 5,716,826, 5,716,613 and U.S. Pat. Pub.No. 20110212530; vaccinia viruses, see, e.g., 2016/0339066, andadeno-associated viruses, see, e.g., U.S. Pat. Nos. 8,007,780,7,968,340, 7,943,374, 7,906,111, 7,927,585, 7,811,814, 7,662,627,7,241,447, 7,238,526, 7,172,893, 7,033,826, 7,001,765, 6,897,045, and6,632,670.

Oncolytic viruses also include viruses that have been geneticallyaltered to attenuate their virulence, to improve their safety profile,enhance their tumor specificity, and they have also been equipped withadditional genes, for example cytotoxins, cytokines, prodrug convertingenzymes to improve the overall efficacy of the viruses (see, e.g., Kirnet al., (2009) Nat Rev Cancer 9:64-71; Garcia-Aragoncillo et al., (2010)Curr Opin Mol Ther 12:403-411; see U.S. Pat. Nos. 7,588,767, 7,588,771,7,662,398 and 7,754,221 and U.S. Pat. Publ. Nos. 2007/0202572,2007/0212727, 2010/0062016, 2009/0098529, 2009/0053244, 2009/0155287,2009/0117034, 2010/0233078, 2009/0162288, 2010/0196325, 2009/0136917 and2011/0064650). In some embodiments, the oncolytic viruses can be thosethat have been modified so that they selectively replicate in cancerouscells, and, thus, are oncolytic. For example, the oncolytic virus is anadenovirus that has been engineered to have modified tropism for tumortherapy and also as gene therapy vectors. Exemplary of such is ONYX-015,H101 and Ad5ΔCR (Hallden and Portella (2012) Expert Opin Ther Targets,16:945-58) and TNFerade (McLoughlin et al. (2005) Ann. Surg. Oncol.,12:825-30), or a conditionally replicative adenovirus Oncorine®.

In some embodiments, the infectious agent is a modified herpes simplexvirus. In some embodiments, the infectious agent is a modified versionof Talimogene laherparepvec (also known as T-Vec, Imlygic or OncoVexGM-CSF), that is modified to contain nucleic acids encoding any of thevariant ICOSL polypeptides or immunomodulatory polypeptides describedherein. In some embodiments, the infectious agent is a modified herpessimplex virus that is described, e.g., in WO 2007/052029, WO1999/038955, US 2004/0063094, US 2014/0154216, or, variants thereof.

In some embodiments, the infectious agent is a virus that targets aparticular type of cells in a subject that is administered the virus,e.g., a virus that targets immune cells or antigen-presenting cells(APCs). Dendritic cells (DCs) are essential APCs for the initiation andcontrol of immune responses. DCs can capture and process antigens,migrate from the periphery to a lymphoid organ, and present the antigensto resting T cells in a major histocompatibility complex(MHC)-restricted fashion. In some embodiments, the infectious agent is avirus that specifically can target DCs to deliver nucleic acids encodingthe variant ICOSL polypeptide or immunomodulatory polypeptides forexpression in DCs. In some embodiments, the virus is a lentivirus or avariant or derivative thereof, such as an integration-deficientlentiviral vector. In some embodiments, the virus is a lentivirus thatis pseudotyped to efficiently bind to and productively infect cellsexpressing the cell surface marker dendritic cell-specific intercellularadhesion molecule-3-grabbing non-integrin (DC-SIGN), such as DCs. Insome embodiments, the virus is a lentivirus pseudotyped with a Sindbisvirus E2 glycoprotein or modified form thereof, such as those describedin WO 2013/149167. In some embodiments, the virus allows for deliveryand expression of a sequence of interest (e.g., a nucleic acid encodingany of the variant ICOSL polypeptides or immunomodulatory polypeptidesdescribed herein) to a DC. In some embodiments, the virus includes thosedescribed in WO 2008/011636, US 2011/0064763, Tareen et al. (2014) Mol.Ther., 22:575-587, or variants thereof. Exemplary of a dendriticcell-tropic vector platform is ZVex™

2. Bacteria

In some embodiments, the infectious agent is a bacterium. For example,in some embodiments, the bacteria can deliver nucleic acids encoding anyof the variant immunomodulatory polypeptides described herein, e.g.,variant ICOSL polypeptide or immunomodulatory polypeptide, to a targetcell in the subject, such as a tumor cell, an immune cell, anantigen-presenting cell and/or a phagocytic cell. In some embodiments,the bacterium can be preferentially targeted to a specific environmentwithin a subject, such as a tumor microenvironment (TME), for expressionand/or secretion of the variant immunomodulatory polypeptides and/or totarget specific cells in the environment for expression of the variantimmunomodulatory polypeptides.

In some embodiments, the bacterium delivers the nucleic acids to thecells via bacterial-mediated transfer of plasmid DNA to mammalian cells(also referred to as “bactofection”). For example, in some embodiments,delivery of genetic material is achieved through entry of the entirebacterium into target cells. In some embodiments, spontaneous or inducedbacterial lysis can lead to the release of plasmid for subsequenteukaryotic cell expression. In some embodiments, the bacterium candeliver nucleic acids to non-phagocytic mammalian cells (e.g., tumorcells) and/or to phagocytic cells, e.g., certain immune cells and/orAPCs. In some embodiments, the nucleic acids delivered by the bacteriumcan be transferred to the nucleus of the cell in the subject forexpression. In some embodiments, the nucleic acids also includeappropriate nucleic acid sequences necessary for the expression of theoperably linked sequences encoding the variant immunomodulatorypolypeptides in a particular host cell, e.g., regulatory elements suchas promoters or enhancers. In some embodiments, the infectious agentthat is a bacterium can deliver nucleic acids encoding theimmunomodulatory proteins in the form of an RNA, such as a pre-madetranslation-competent RNA delivered to the cytoplasm of the target cellfor translation by the target cell's machinery.

In some embodiments, the bacterium can replicate and lyse the targetcells, e.g., tumor cells. In some embodiments, the bacterium can containand/or release nucleic acid sequences and/or gene products in thecytoplasm of the target cells, thereby killing the target cell, e.g.,tumor cell. In some embodiments, the infectious agent is bacterium thatcan replicate specifically in a particular environment in the subject,e.g., tumor microenvironment (TME). For example, in some embodiments,the bacterium can replicate specifically in anaerobic or hypoxicmicroenvironments. In some embodiments, conditions or factors present inparticular environments, e.g., aspartate, serine, citrate, ribose orgalactose produced by cells in the TME, can act as chemoattractants toattract the bacterium to the environment. In some embodiments, thebacterium can express and/or secrete the immunomodulatory proteinsdescribed herein in the environment, e.g., TME.

In some embodiments, the infectious agent is a bacterium that is aListeria sp., a Bifidobacterium sp., an Escherichia sp., a Clostridiumsp., a Salmonella sp., a Shigella sp., a Vibrio sp. or a Yersinia sp. Insome embodiments, the bacterium is selected from among one or more ofListeria monocytogenes, Salmonella typhimurium, Salmonella choleraesuis,Escherichia coli, Vibrio cholera, Clostridium perfringens, Clostridiumbutyricum, Clostridium novyi, Clostridium acetobutylicum,Bifidobacterium infantis, Bifidobacterium longum and Bifidobacteriumadolescentis. In some embodiments, the bacterium is an engineeredbacterium. In some embodiments, the bacterium is an engineered bacteriumsuch as those described in, e.g., Seow and Wood (2009) Molecular Therapy17(5):767-777; Baban et al. (2010) Bioengineered Bugs 1:6, 385-394;Patyar et al. (2010) J Biomed Sci 17:21; Tangney et al. (2010)Bioengineered Bugs 1:4, 284-287; van Pijkeren et al. (2010) Hum GeneTher. 21(4):405-416; WO 2012/149364; WO 2014/198002; U.S. Pat. Nos.9,103,831; 9,453,227; US 2014/0186401; US 2004/0146488; US 2011/0293705;US 2015/0359909 and EP 3020816. The bacterium can be modified to delivernucleic acid sequences encoding any of the variant immunomodulatorypolypeptides, conjugates and/or fusions provided herein, and/or toexpress such variant immunomodulatory polypeptides in the subject.

IV. NUCLEIC ACIDS, VECTORS AND METHODS FOR PRODUCING THE POLYPEPTIDES ORCELLS

Provided herein are isolated or recombinant nucleic acids collectivelyreferred to as “nucleic acids” which encode any of the various providedembodiments of the variant ICOSL polypeptides or immunomodulatorypolypeptides provided herein. In some embodiments, nucleic acidsprovided herein, including all described below, are useful inrecombinant production (e.g., expression) of variant ICOSL polypeptidesor immunomodulatory polypeptides provided herein. In some embodiments,nucleic acids provided herein, including all described below, are usefulin expression of variant ICOSL polypeptides or immunomodulatorypolypeptides provided herein in cells, such as in engineered cells, e.g.immune cells, or infectious agent cells. The nucleic acids providedherein can be in the form of RNA or in the form of DNA, and includemRNA, cRNA, recombinant or synthetic RNA and DNA, and cDNA. The nucleicacids provided herein are typically DNA molecules, and usuallydouble-stranded DNA molecules. However, single-stranded DNA,single-stranded RNA, double-stranded RNA, and hybrid DNA/RNA nucleicacids or combinations thereof comprising any of the nucleotide sequencesof the invention also are provided.

Also provided herein are recombinant expression vectors and recombinanthost cells useful in producing the variant ICOSL polypeptides orimmunomodulatory polypeptides provided herein.

Also provided herein are engineered cells, such as engineered immunecells, containing any of the provided nucleic acid molecules or any ofthe variant ICOSL polypeptides or immunomodulatory polypeptides, such asany of the transmembrane immunomodulatory polypeptides or secretableimmunomodulatory polypeptides.

Also provided herein are infectious agents, such as bacterial or viralcells, containing any of the provided nucleic acid molecules or any ofthe variant ICOSL polypeptides or immunomodulatory polypeptides, such asany of the transmembrane immunomodulatory polypeptides or secretableimmunomodulatory polypeptides.

In any of the above provided embodiments, the nucleic acids encoding thevariant polypeptides or immunomodulatory polypeptides provided hereincan be introduced into cells using recombinant DNA and cloningtechniques. To do so, a recombinant DNA molecule encoding animmunomodulatory polypeptide is prepared. Methods of preparing such DNAmolecules are well known in the art. For instance, sequences coding forthe peptides could be excised from DNA using suitable restrictionenzymes. Alternatively, the DNA molecule could be synthesized usingchemical synthesis techniques, such as the phosphoramidite method. Also,a combination of these techniques could be used. In some instances, arecombinant or synthetic nucleic acid may be generated throughpolymerase chain reaction (PCR). In some embodiments, a DNA insert canbe generated encoding one or more variant ICOSL polypeptides containingat least one affinity-modified IgSF domain and, in some embodiments, asignal peptide, a transmembrane domain and/or an endodomain in accordwith the provided description. This DNA insert can be cloned into anappropriate transduction/transfection vector as is known to those ofskill in the art. Also provided are expression vectors containing thenucleic acid molecules.

In some embodiments, the expression vectors are capable of expressingthe immunomodulatory proteins in an appropriate cell under conditionssuited to expression of the protein. In some aspects, nucleic acidmolecule or an expression vector comprises the DNA molecule that encodesthe immunomodulatory protein operatively linked to appropriateexpression control sequences. Methods of effecting this operativelinking, either before or after the DNA molecule is inserted into thevector, are well known. Expression control sequences include promoters,activators, enhancers, operators, ribosomal binding sites, startsignals, stop signals, cap signals, polyadenylation signals, and othersignals involved with the control of transcription or translation.

In some embodiments, expression of the immunomodulatory protein iscontrolled by a promoter or enhancer to control or regulate expression.The promoter is operably linked to the portion of the nucleic acidmolecule encoding the variant polypeptide or immunomodulatory protein.In some embodiments, the promotor is a constitutively active promotor(such as a tissue-specific constitutively active promotor or otherconstitutive promotor). In some embodiments, the promotor is aninducible promotor, which may be responsive to an inducing agent (suchas a T cell activation signal).

In some embodiments, a constitutive promoter is operatively linked tothe nucleic acid molecule encoding the variant polypeptide orimmunomodulatory protein. Exemplary constitutive promoters include theSimian vacuolating virus 40 (SV40) promoter, the cytomegalovirus (CMV)promoter, the ubiquitin C (UbC) promoter, and the EF-1 alpha (EF1a)promoter. In some embodiments, the constitutive promoter is tissuespecific. For example, in some embodiments, the promoter allows forconstitutive expression of the immunomodulatory protein in specifictissues, such as immune cells, lymphocytes, or T cells. Exemplarytissue-specific promoters are described in U.S. Pat. No. 5,998,205,including, for example, a fetoprotein, DF3, tyrosinase, CEA, surfactantprotein, and ErbB2 promoters.

In some embodiments, an inducible promoter is operatively linked to thenucleic acid molecule encoding the variant polypeptide orimmunomodulatory protein such that expression of the nucleic acid iscontrollable by controlling the presence or absence of the appropriateinducer of transcription. For example, the promoter can be a regulatedpromoter and transcription factor expression system, such as thepublished tetracycline-regulated systems or other regulatable systems(see, e.g. published International PCT Appl. No. WO 01/30843), to allowregulated expression of the encoded polypeptide. An exemplaryregulatable promoter system is the Tet-On (and Tet-Off) systemavailable, for example, from Clontech (Palo Alto, Calif.). This promotersystem allows the regulated expression of the transgene controlled bytetracycline or tetracycline derivatives, such as doxycycline. Otherregulatable promoter systems are known (see e.g., published U.S.Application No. 2002-0168714, entitled “Regulation of Gene ExpressionUsing Single-Chain, Monomeric, Ligand Dependent Polypeptide Switches,”which describes gene switches that contain ligand binding domains andtranscriptional regulating domains, such as those from hormonereceptors).

In some embodiments, the promotor is responsive to an element responsiveto T-cell activation signaling. Solely by way of example, in someembodiments, an engineered T cell comprises an expression vectorencoding the immunomodulatory protein and a promotor operatively linkedto control expression of the immunomodulatory protein. The engineered Tcell can be activated, for example by signaling through an engineered Tcell receptor (TCR) or a chimeric antigen rector (CAR), and therebytriggering expression and secretion of the immunomodulatory proteinthrough the responsive promotor.

In some embodiments, an inducible promoter is operatively linked to thenucleic acid molecule encoding the immunomodulatory protein such thatthe immunomodulatory protein is expressed in response to a nuclearfactor of activated T-cells (NFAT) or nuclear factor kappa-light-chainenhancer of activated B cells (NF-κB). For example, in some embodiments,the inducible promoter comprises a binding site for NFAT or NF-κB. Forexample, in some embodiments, the promoter is an NFAT or NF-κB promoteror a functional variant thereof. Thus, in some embodiments, the nucleicacids make it possible to control the expression of immunomodulatoryprotein while also reducing or eliminating the toxicity of theimmunomodulatory protein. In particular, engineered immune cellscomprising the nucleic acids of the invention express and secrete theimmunomodulatory protein only when the cell (e.g., a T-cell receptor(TCR) or a chimeric antigen receptor (CAR) expressed by the cell) isspecifically stimulated by an antigen and/or the cell (e.g., the calciumsignaling pathway of the cell) is non-specifically stimulated by, e.g.,phorbol myristate acetate (PMA)/Ionomycin. Accordingly, the expressionand, in some cases, secretion, of immunomodulatory protein can becontrolled to occur only when and where it is needed (e.g., in thepresence of an infectious disease-causing agent, cancer, or at a tumorsite), which can decrease or avoid undesired immunomodulatory proteininteractions.

In some embodiments, the nucleic acid encoding an immunomodulatoryprotein described herein comprises a suitable nucleotide sequence thatencodes a NFAT promoter, NF-κB promoter, or a functional variantthereof. “NFAT promoter” as used herein means one or more NFATresponsive elements linked to a minimal promoter. “NF-κB promoter”refers to one or more NF-κB responsive elements linked to a minimalpromoter. In some embodiments, the minimal promoter of a gene is aminimal human IL-2 promoter or a CMV promoter. The NFAT responsiveelements may comprise, e.g., NFAT1, NFAT2, NFAT3, and/or NFAT4responsive elements. The NFAT promoter, NF-κB promoter, or a functionalvariant thereof may comprise any number of binding motifs, e.g., atleast two, at least three, at least four, at least five, or at leastsix, at least seven, at least eight, at least nine, at least ten, atleast eleven, or up to twelve binding motifs.

The resulting recombinant expression vector having the DNA moleculethereon is used to transform an appropriate host. This transformationcan be performed using methods well known in the art. In someembodiments, a nucleic acid provided herein further comprises nucleotidesequence that encodes a secretory or signal peptide operably linked tothe nucleic acid encoding an immunomodulatory polypeptide such that aresultant soluble immunomodulatory polypeptide is recovered from theculture medium, host cell, or host cell periplasm. In other embodiments,the appropriate expression control signals are chosen to allow formembrane expression of an immunomodulatory polypeptide. Furthermore,commercially available kits as well as contract manufacturing companiescan also be utilized to make engineered cells or recombinant host cellsprovided herein.

In some embodiments, the resulting expression vector having the DNAmolecule thereon is used to transform, such as transduce, an appropriatecell. The introduction can be performed using methods well known in theart. Exemplary methods include those for transfer of nucleic acidsencoding the receptors, including via viral, e.g., retroviral orlentiviral, transduction, transposons, and electroporation. In someembodiments, the expression vector is a viral vector. In someembodiments, the nucleic acid is transferred into cells by lentiviral orretroviral transduction methods.

Any of a large number of publicly available and well-known mammalianhost cells, including mammalian T-cells or APCs, can be used in thepreparing the polypeptides or engineered cells. The selection of a cellis dependent upon a number of factors recognized by the art. Theseinclude, for example, compatibility with the chosen expression vector,toxicity of the peptides encoded by the DNA molecule, rate oftransformation, ease of recovery of the peptides, expressioncharacteristics, bio-safety and costs. A balance of these factors mustbe struck with the understanding that not all cells can be equallyeffective for the expression of a particular DNA sequence.

In some embodiments, the host cells can be a variety of eukaryoticcells, such as in yeast cells, or with mammalian cells such as Chinesehamster ovary (CHO) or HEK293 cells. In some embodiments, the host cellis a suspension cell and the polypeptide is engineered or produced incultured suspension, such as in cultured suspension CHO cells, e.g.CHO-S cells. In some examples, the cell line is a CHO cell line that isdeficient in DHFR (DHFR−), such as DG44 and DUXB11. In some embodiments,the cell is deficient in glutamine synthase (GS), e.g. CHO-S cells,CHOK1 SV cells, and CHOZN((R)) GS−/− cells. In some embodiments, the CHOcells, such as suspension CHO cells, may be CHO-S-2H2 cells, CHO-S-clone14 cells, or ExpiCHO-S cells.

In some embodiments, expressing the provided ICOSL polypeptides from CHOcells results in a more homogenous composition of produced proteins. Insome embodiments, the provided ICOSL polypeptides results in a morehomogenous product when the proteins are expressed from CHO cellscompared to ICOSL polypeptides containing the full ECD referencesequence and/or containing the protease cleavage site (e.g., LQQN/LT).In some embodiments, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% of the composition of produced proteins containing an ICOSL variantpolypeptide produced herein, have the same amino acid length or are thesame size. Techniques to assess homogeneity of size include highperformance liquid chromatography (HPLC), size exclusion chromatography,SDS page, or sequencing.

In some embodiments, host cells can also be prokaryotic cells, such aswith E. coli. The transformed recombinant host is cultured underpolypeptide expressing conditions, and then purified to obtain a solubleprotein. Recombinant host cells can be cultured under conventionalfermentation conditions so that the desired polypeptides are expressed.Such fermentation conditions are well known in the art. Finally, thepolypeptides provided herein can be recovered and purified fromrecombinant cell cultures by any of a number of methods well known inthe art, including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, and affinitychromatography. Protein refolding steps can be used, as desired, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed in the finalpurification steps.

In some embodiments, the cell is an immune cell, such as any describedabove in connection with preparing engineered cells. In someembodiments, such engineered cells are primary cells. In someembodiments, the engineered cells are autologous to the subject. In someembodiment, the engineered cells are allogeneic to the subject. In someembodiments, the engineered cells are obtained from a subject, such asby leukapheresis, and transformed ex vivo for expression of theimmunomodulatory polypeptide, e.g. transmembrane immunomodulatorypolypeptide or secretable immunomodulatory polypeptide.

Also provided are nucleic acids encoding any of the variantimmunomodulatory polypeptides contained in infectious agents describedherein. In some embodiments, the infectious agents deliver the nucleicacids to a cell in the subject, and/or permit expression of the encodedvariant polypeptides in the cell. Also provided are nucleic acids thatare used to generate, produce or modify such infectious agents. Forexample, in some embodiments, provided are vectors and/or plasmids thatcontain nucleic acids encoding the variant immunomodulatorypolypeptides, for generation of the infectious agents, delivery to thecells in a subject and/or expression of the variant immunomodulatorypolypeptides in the cells in the subject.

In some embodiments, the provided nucleic acids are recombinant viral orbacterial vectors containing nucleic acid sequences encoding the variantimmunomodulatory polypeptides. In some embodiments, the recombinantvectors can be used to produce an infectious agent that contains nucleicacid sequences encoding the variant immunomodulatory polypeptides and/orto be delivered to a target cell in the subject for expression by thetarget cell. In some embodiments, the recombinant vector is anexpression vector. In some embodiments, the recombinant vector includesappropriate sequences necessary for generation and/or production of theinfectious agent and expression in the target cell.

In some embodiments, the recombinant vector is a plasmid or cosmid.Plasmid or cosmid containing nucleic acid sequences encoding the variantimmunomodulatory polypeptides, as described herein, is readilyconstructed using standard techniques well known in the art. Forgeneration of the infectious agent, the vector or genome can beconstructed in a plasmid form that can then be transfected into apackaging or producer cell line or a host bacterium. The recombinantvectors can be generated using any of the recombinant techniques knownin the art. In some embodiments, the vectors can include a prokaryoticorigin of replication and/or a gene whose expression confers adetectable or selectable marker such as a drug resistance forpropagation and/or selection in prokaryotic systems.

In some embodiments, the recombinant vector is a viral vector. Exemplaryrecombinant viral vectors include a lentiviral vector genome, poxvirusvector genome, vaccinia virus vector genome, adenovirus vector genome,adenovirus-associated virus vector genome, herpes virus vector genome,and alpha virus vector genome. Viral vectors can be live, attenuated,replication conditional or replication deficient, non-pathogenic(defective), replication competent viral vector, and/or is modified toexpress a heterologous gene product, e.g., the variant immunomodulatorypolypeptides provided herein. Vectors for generation of viruses also canbe modified to alter attenuation of the virus, which includes any methodof increasing or decreasing the transcriptional or translational load.

Exemplary viral vectors that can be used include modified vaccinia virusvectors (see, e.g., Guerra et al., J. Virol. 80:985-98 (2006); Tartagliaet al., AIDS Research and Human Retroviruses 8: 1445-47 (1992); Gheradiet al., J. Gen. Virol. 86:2925-36 (2005); Mayr et al., Infection 3:6-14(1975); Hu et al., J. Virol. 75: 10300-308 (2001); U.S. Pat. Nos.5,698,530, 6,998,252, 5,443,964, 7,247,615 and 7,368,116); adenovirusvector or adenovirus-associated virus vectors (see., e.g., Molin et al.,J. Virol. 72:8358-61 (1998); Narumi et al., Am J. Respir. Cell Mol.Biol. 19:936-41 (1998); Mercier et al., Proc. Natl. Acad. Sci. USA101:6188-93 (2004); U.S. Pat. Nos. 6,143,290; 6,596,535; 6,855,317;6,936,257; 7,125,717; 7,378,087; 7,550,296); retroviral vectorsincluding those based upon murine leukemia virus (MuLV), gibbon apeleukemia virus (GaLV), ecotropic retroviruses, simian immunodeficiencyvirus (SIV), human immunodeficiency virus (HIV), and combinations (see,e.g., Buchscher et al., J. Virol. 66:2731-39 (1992); Johann et al., J.Virol. 66: 1635-40 (1992); Sommerfelt et al., Virology 176:58-59 (1990);Wilson et al., J. Virol. 63:2374-78 (1989); Miller et al., J. Virol.65:2220-24 (1991); Miller et al., Mol. Cell Biol. 10:4239 (1990);Kolberg, NIH Res. 4:43 1992; Cornetta et al., Hum. Gene Ther. 2:215(1991)); lentiviral vectors including those based upon HumanImmunodeficiency Virus (HIV-1), HIV-2, feline immunodeficiency virus(FIV), equine infectious anemia virus, Simian Immunodeficiency Virus(SIV), and maedi/visna virus (see, e.g., Pfeifer et al., Annu. Rev.Genomics Hum. Genet. 2: 177-211 (2001); Zufferey et al., J. Virol. 72:9873, 1998; Miyoshi et al., J. Virol. 72:8150, 1998; Philpott andThrasher, Human Gene Therapy 18:483, 2007; Engelman et al., J. Virol.69: 2729, 1995; Nightingale et al., Mol. Therapy, 13: 1121, 2006; Brownet al., J. Virol. 73:9011 (1999); WO 2009/076524; WO 2012/141984; WO2016/011083; McWilliams et al., J. Virol. 77: 11150, 2003; Powell etal., J. Virol. 70:5288, 1996) or any, variants thereof, and/or vectorsthat can be used to generate any of the viruses described above. In someembodiments, the recombinant vector can include regulatory sequences,such as promoter or enhancer sequences, that can regulate the expressionof the viral genome, such as in the case for RNA viruses, in thepackaging cell line (see, e.g., U.S. Pat. Nos. 5,385,839 and 5,168,062).

In some embodiments, the recombinant vector is an expression vector,e.g., an expression vector that permits expression of the encoded geneproduct when delivered into the target cell, e.g., a cell in thesubject, e.g., a tumor cell, an immune cell and/or an APC. In someembodiments, the recombinant expression vectors contained in theinfectious agent are capable of expressing the immunomodulatory proteinsin the target cell in the subject, under conditions suited to expressionof the protein.

In some aspects, nucleic acids or an expression vector comprises anucleic acid sequence that encodes the immunomodulatory proteinoperatively linked to appropriate expression control sequences. Methodsof affecting this operative linking, either before or after the nucleicacid sequence encoding the immunomodulatory protein is inserted into thevector, are well known. Expression control sequences include promoters,activators, enhancers, operators, ribosomal binding sites, startsignals, stop signals, cap signals, polyadenylation signals, and othersignals involved with the control of transcription or translation. Thepromoter can be operably linked to the portion of the nucleic acidsequence encoding the immunomodulatory protein. In some embodiments, thepromotor is a constitutively active promotor in the target cell (such asa tissue-specific constitutively active promotor or other constitutivepromotor). For example, the recombinant expression vector may alsoinclude, lymphoid tissue-specific transcriptional regulatory elements(TRE) such as a B lymphocyte, T lymphocyte, or dendritic cell specificTRE. Lymphoid tissue specific TRE are known in the art (see, e.g.,Thompson et al., Mol. Cell. Biol. 12:1043-53 (1992); Todd et al., J.Exp. Med. 177:1663-74 (1993); Penix et al., J. Exp. Med. 178:1483-96(1993)). In some embodiments, the promotor is an inducible promotor,which may be responsive to an inducing agent (such as a T cellactivation signal). In some embodiments, nucleic acids delivered to thetarget cell in the subject, e.g., tumor cell, immune cell and/or APC,can be operably linked to any of the regulatory elements describedabove.

In some embodiments, the vector is a bacterial vector, e.g., a bacterialplasmid or cosmid. In some embodiments, the bacterial vector isdelivered to the target cell, e.g., tumor cells, immune cells and/orAPCs, via bacterial-mediated transfer of plasmid DNA to mammalian cells(also referred to as “bactofection”). In some embodiments, the deliveredbacterial vector also contains appropriate expression control sequencesfor expression in the target cells, such as a promoter sequence and/orenhancer sequences, or any regulatory or control sequences describedabove. In some embodiments, the bacterial vector contains appropriateexpression control sequences for expression and/or secretion of theencoded variant polypeptides in the infectious agent, e.g., thebacterium.

In some embodiments, polypeptides provided herein can also be made bysynthetic methods. Solid phase synthesis is the preferred technique ofmaking individual peptides since it is the most cost-effective method ofmaking small peptides. For example, well known solid phase synthesistechniques include the use of protecting groups, linkers, and solidphase supports, as well as specific protection and deprotection reactionconditions, linker cleavage conditions, use of scavengers, and otheraspects of solid phase peptide synthesis. Peptides can then be assembledinto the polypeptides as provided herein.

V. METHODS OF ASSESSING ACTIVITY IMMUNE MODULATION OF VARIANT ICOSLPOLYPEPTIDES AND IMMUNOMODULATORY PROTEINS

In some embodiments, the variant ICOSL polypeptides provided herein(e.g. full-length and/or specific binding fragments or conjugates, stackconstructs or fusion thereof) exhibit immunomodulatory activity tomodulate T cell activation. In some embodiments, ICOSL polypeptidesmodulate IFN-gamma expression in a primary T cell assay relative to areference (e.g., unmodified) or wild-type ICOSL control. In some cases,modulation of IFN-gamma expression can increase or decrease IFN-gammaexpression relative to the control. Assays to determine specific bindingand IFN-gamma expression are well-known in the art and include the MLR(mixed lymphocyte reaction) assays measuring interferon-gamma cytokinelevels in culture supernatants (Wang et al., Cancer Immunol Res. 2014September: 2(9):846-56), SEB (staphylococcal enterotoxin B) T cellstimulation assay (Wang et al., Cancer Immunol Res. 2014 September:2(9):846-56), and anti-CD3 T cell stimulation assays (Li and Kurlander,J Transl Med. 2010: 8: 104).

In some embodiments, a variant ICOSL polypeptide can in some embodimentsincrease or, in alternative embodiments, decrease IFN-gamma(interferon-gamma) expression in a primary T-cell assay relative to awild-type ICOSL control. In some embodiments of the providedpolypeptides containing a soluble variant ICOSL sequence, thepolypeptide can increase IFN-gamma expression and, in alternativeembodiments, decrease IFN-gamma expression in a primary T-cell assayrelative to a wild-type ICOSL control. In some embodiments of theprovided polypeptides containing multiple variant ICOSL sequences, thepolypeptide can increase IFN-gamma expression and, in alternativeembodiments, decrease IFN-gamma expression in a primary T-cell assayrelative to a wild-type ICOSL control.

Those of skill will recognize that the format of the primary T-cellassay used to determine an increase in IFN-gamma expression can differfrom that employed to assay for a decrease in IFN-gamma expression. Inassaying for the ability of a variant ICOSL to decrease IFN-gammaexpression in a primary T-cell assay, a Mixed Lymphocyte Reaction (MLR)assay can be used as described in Example 6. In some cases, a solubleform of a variant ICOSL can be employed to determine the ability of thevariant ICOSL to antagonize and thereby decrease the IFN-gammaexpression in a MLR as likewise described in Example 6.

Alternatively, in assaying for the ability of a variant ICOSL toincrease IFN-gamma expression in a primary T-cell assay, aco-immobilization assay can be used as described in Example 6. In aco-immobilization assay, a TCR signal, provided in some embodiments byanti-CD3 antibody, is used in conjunction with a co-immobilized variantICOSL to determine the ability to increase IFN-gamma expression relativeto an ICOSL control. In some cases, a soluble form of a variant ICOSLthat is multimerized to a degree to provide multivalent binding can beemployed to determine the ability of the variant ICOSL to agonize andthereby increase the IFN-gamma expression in a MLR as likewise describedin Example 6.

In some embodiments, in assaying for the ability of a variant ICOSL tomodulate an increase or decrease IFN-gamma expression a T cell reporterassay can be used. In some embodiments, the T cell is a Jurkat T cellline or is derived from Jurkat T cell lines. In reporter assays, thereporter cell line (e.g. Jurkat reporter cell) also is generated tooverexpress an inhibitory receptor that is the cognate binding partnerof the variant IgSF domain polypeptide. In some embodiments, thereporter T cells also contain a reporter construct containing aninducible promoter responsive to T cell activation operably linked to areporter. In some embodiments, the reporter is a fluorescent orluminescent reporter. In some embodiments, the reporter is luciferase.In some embodiments, the promoter is responsive to CD3 signaling. Insome embodiments, the promoter is an NFAT promoter. In some embodiments,the promoter is responsive to costimulatory signaling, e.g. CD28costimulatory signaling. In some embodiments, the promoter is an IL-2promoter.

In aspects of a reporter assay, a reporter cell line is stimulated, suchas by co-incubation with antigen presenting cells (APCs) expressing thewild-type ligand of the inhibitory receptor, e.g. ICOSL. In someembodiments, the APCs are artificial APCs. Artificial APCs are wellknown to a skilled artisan. In some embodiments, artificial APCs arederived from one or more mammalian cell line, such as K562, CHO or 293cells.

In some embodiments, the Jurkat reporter cells are co-incubated withartificial APCs overexpressing the inhibitory ligand in the presence ofthe variant IgSF domain molecule or immunomodulatory protein, e.g.,variant ICOSL polypeptide or immunomodulatory protein. In someembodiments, reporter expression is monitored, such as by determiningthe luminescence or fluorescence of the cells. In some embodiments,normal interactions between its inhibitory receptor and ligand result ina repression of or decrease in the reporter signal, such as compared tocontrol, e.g. reporter expression by co-incubation of control T cellsand APCs in which the inhibitory receptor and ligand interaction is notpresent, e.g. APCs that do not overexpress ICOSL. In some embodiments, avariant ICOSL polypeptide or immunomodulatory protein provided hereinantagonizes the interaction, e.g. when provided in soluble form as avariant ICOSL-Fc or when expressed from the APC as a secretableimmunomodulatory protein, thereby resulting in an increase in thereporter signal compared to the absence of the variant ICOSL polypeptideor immunomodulatory protein. In some cases, certain formats of a variantICOSL polypeptide or immunomodulatory protein as provided herein mayprovide an agonist activity, thereby decreasing reporter expressioncompared to the absence of the variant ICOSL polypeptide orimmunomodulatory protein.

Use of proper controls is known to those of skill in the art, however,in the aforementioned embodiments, the control typically involves use ofthe reference ICOSL, such as a wild-type of native ICOSL isoform fromthe same mammalian species from which the variant ICOSL was derived ordeveloped. Irrespective of whether the binding affinity to either one orboth of ICOS and CD28 is increased or decreased, a variant ICOSL in someembodiments will increase IFN-gamma expression and, in alternativeembodiments, decrease IFN-gamma expression in a primary T-cell assayrelative to a wild-type ICOSL control.

In some embodiments, a variant ICOSL increases IFN-gamma expression(i.e., protein expression) relative to a reference (e.g., unmodified) orwild-type ICOSL control by at least: 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or higher. In other embodiments, a variant ICOSLdecreases IFN-gamma expression (i.e. protein expression) relative to awild-type or unmodified ICOSL control by at least: 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or higher. In some embodiments, thewild-type ICOSL control is murine ICOSL, such as would typically be usedfor a variant ICOSL altered in sequence from that of a wild-type murineICOSL sequence. In some embodiments, the wild-type ICOSL control ishuman ICOSL, such as would typically be used for a variant ICOSL alteredin sequence from that of a wild-type human ICOSL sequence such as anICOSL sequence comprising the sequence of amino acids of SEQ ID NO:32 orSEQ ID NO:196 or 545.

VI. PHARMACEUTICAL FORMULATIONS

Provided herein are compositions containing any of the variant ICOSLpolypeptides, immunodulatory proteins, conjugates, engineered cells orinfectious agents described herein. The pharmaceutical composition canfurther comprise a pharmaceutically acceptable excipient. For example,the pharmaceutical composition can contain one or more excipients formodifying, maintaining or preserving, for example, the pH, osmolarity,viscosity, clarity, color, isotonicity, odor, sterility, stability, rateof dissolution or release, adsorption, or penetration of thecomposition. In some aspects, a skilled artisan understands that apharmaceutical composition containing cells may differ from apharmaceutical composition containing a protein.

In some embodiments, the pharmaceutical composition is a solid, such asa powder, capsule, or tablet. For example, the components of thepharmaceutical composition can be lyophilized. In some embodiments, thesolid pharmaceutical composition is reconstituted or dissolved in aliquid prior to administration.

In some embodiments, the pharmaceutical composition is a liquid, forexample variant ICOSL polypeptides dissolved in an aqueous solution(such as physiological saline or Ringer's solution). In someembodiments, the pH of the pharmaceutical composition is between about4.0 and about 8.5 (such as between about 4.0 and about 5.0, betweenabout 4.5 and about 5.5, between about 5.0 and about 6.0, between about5.5 and about 6.5, between about 6.0 and about 7.0, between about 6.5and about 7.5, between about 7.0 and about 8.0, or between about 7.5 andabout 8.5).

In some embodiments, the pharmaceutical composition comprises apharmaceutically-acceptable excipient, for example a filler, binder,coating, preservative, lubricant, flavoring agent, sweetening agent,coloring agent, a solvent, a buffering agent, a chelating agent, orstabilizer. Examples of pharmaceutically-acceptable fillers includecellulose, dibasic calcium phosphate, calcium carbonate,microcrystalline cellulose, sucrose, lactose, glucose, mannitol,sorbitol, maltol, pregelatinized starch, corn starch, or potato starch.Examples of pharmaceutically-acceptable binders includepolyvinylpyrrolidone, starch, lactose, xylitol, sorbitol, maltitol,gelatin, sucrose, polyethylene glycol, methyl cellulose, or cellulose.Examples of pharmaceutically-acceptable coatings include hydroxypropylmethylcellulose (HPMC), shellac, corn protein zein, or gelatin. Examplesof pharmaceutically-acceptable disintegrants includepolyvinylpyrrolidone, carboxymethyl cellulose, or sodium starchglycolate. Examples of pharmaceutically-acceptable lubricants includepolyethylene glycol, magnesium stearate, or stearic acid. Examples ofpharmaceutically-acceptable preservatives include methyl parabens, ethylparabens, propyl paraben, benzoic acid, or sorbic acid. Examples ofpharmaceutically-acceptable sweetening agents include sucrose,saccharine, aspartame, or sorbitol. Examples ofpharmaceutically-acceptable buffering agents include carbonates,citrates, gluconates, acetates, phosphates, or tartrates.

In some embodiments, the pharmaceutical composition further comprises anagent for the controlled or sustained release of the product, such asinjectable microspheres, bio-erodible particles, polymeric compounds(polylactic acid, polyglycolic acid), beads, or liposomes.

In some embodiments, the pharmaceutical composition is sterile.Sterilization may be accomplished by filtration through sterilefiltration membranes or radiation. Where the composition is lyophilized,sterilization using this method may be conducted either prior to orfollowing lyophilization and reconstitution. The composition forparenteral administration may be stored in lyophilized form or insolution. In addition, parenteral compositions generally are placed intoa container having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

In some embodiments, provided are pharmaceutical compositions containingthe transmembrane immunomodulatory proteins, including engineered cellsexpressing such transmembrane immunomodulatory proteins. In someembodiments, the pharmaceutical compositions and formulations includeone or more optional pharmaceutically acceptable carrier or excipient.Such compositions may comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.Compositions of the present invention are preferably formulated forintravenous administration.

In some embodiments, the pharmaceutical composition contains infectiousagents containing nucleic acid sequences encoding the immunomodulatoryvariant polypeptides. In some embodiments, the pharmaceuticalcomposition contains a dose of infectious agents suitable foradministration to a subject that is suitable for treatment. In someembodiments, the pharmaceutical composition contains an infectious agentthat is a virus, at a single or multiple dosage amount, of between aboutbetween or between about 1×10⁵ and about 1×10¹² plaque-forming units(pfu), 1×10⁶ and 1×10¹⁰ pfu, or 1×10⁷ and 1×10¹⁰ pfu, each inclusive,such as at least or at least about or at about 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹ pfu or about 1×10¹⁰ pfu. In someembodiments, the pharmaceutical composition can contain a virusconcentration of from or from about 10⁵ to about 10¹⁰ pfu/mL, forexample, 5×10⁶ to 5×10⁹ or 1×10⁷ to 1×10⁹ pfu/mL, such as at least or atleast about or at about 10⁶ pfu/mL, 10⁷ pfu/mL, 10⁸ pfu/mL or 10⁹pfu/mL. In some embodiments, the pharmaceutical composition contains aninfectious agent that is a bacterium, at a single or multiple dosageamount, of between about between or between about 1×10³ and about 1×10⁹colony-forming units (cfu), 1×10⁴ and 1×10⁹ cfu, or 1×10⁵ and 1×10⁷ cfu,each inclusive, such as at least or at least about or at about 1×10⁴,1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸ or 1×10⁹ cfu. In some embodiments, thepharmaceutical composition can contain a bacterial concentration of fromor from about 10³ to about 10⁸ cfu/mL, for example, 5×10⁵ to 5×10⁷ or1×10⁶ to 1×10⁷ cfu/mL, such as at least or at least about or at about10⁵ cfu/mL, 10⁶ cfu/mL, 10⁷ cfu/mL or 10⁸ cfu/mL

Such a formulation may, for example, be in a form suitable forintravenous infusion. A pharmaceutically acceptable carrier may be apharmaceutically acceptable material, composition, or vehicle that isinvolved in carrying or transporting cells of interest from one tissue,organ, or portion of the body to another tissue, organ, or portion ofthe body. For example, the carrier may be a liquid or solid filler,diluent, excipient, solvent, or encapsulating material, or somecombination thereof. Each component of the carrier must be“pharmaceutically acceptable” in that it must be compatible with theother ingredients of the formulation. It also must be suitable forcontact with any tissue, organ, or portion of the body that it mayencounter, meaning that it must not carry a risk of toxicity,irritation, allergic response, immunogenicity, or any other complicationthat excessively outweighs its therapeutic benefits.

In some embodiments, the pharmaceutical composition is administered to asubject. Generally, dosages and routes of administration of thepharmaceutical composition are determined according to the size andcondition of the subject, according to standard pharmaceutical practice.For example, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models such asmice, rats, rabbits, dogs, pigs, or monkeys. An animal model may also beused to determine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans. The exact dosage will bedetermined in light of factors related to the subject requiringtreatment. Dosage and administration are adjusted to provide sufficientlevels of the active compound or to maintain the desired effect. Factorsthat may be taken into account include the severity of the diseasestate, the general health of the subject, the age, weight, and gender ofthe subject, time and frequency of administration, drug combination(s),reaction sensitivities, and response to therapy.

Long-acting pharmaceutical compositions may be administered every 3 to 4days, every week, or biweekly depending on the half-life and clearancerate of the particular formulation. The frequency of dosing will dependupon the pharmacokinetic parameters of the molecule in the formulationused. Typically, a composition is administered until a dosage is reachedthat achieves the desired effect. The composition may therefore beadministered as a single dose, or as multiple doses (at the same ordifferent concentrations/dosages) over time, or as a continuousinfusion. Further refinement of the appropriate dosage is routinelymade. Appropriate dosages may be ascertained through use of appropriatedose-response data. A number of biomarkers or physiological markers fortherapeutic effect can be monitored including T cell activation orproliferation, cytokine synthesis or production (e.g., production ofTNF-α, IFN-γ, IL-2), induction of various activation markers (e.g.,CD25, IL-2 receptor), inflammation, joint swelling or tenderness, serumlevel of C-reactive protein, anti-collagen antibody production, and/or Tcell-dependent antibody response(s).

In some embodiments, the pharmaceutical composition is administered to asubject through any route, including orally, transdermally, byinhalation, intravenously, intra-arterially, intramuscularly, directapplication to a wound site, application to a surgical site,intraperitoneally, by suppository, subcutaneously, intradermally,transcutaneously, by nebulization, intrapleurally, intraventricularly,intra-articularly, intraocularly, or intraspinally.

In some embodiments, the dosage of the pharmaceutical composition is asingle dose or a repeated dose. In some embodiments, the doses are givento a subject once per day, twice per day, three times per day, or fouror more times per day. In some embodiments, about 1 or more (such asabout 2 or more, about 3 or more, about 4 or more, about 5 or more,about 6 or more, or about 7 or more) doses are given in a week. In someembodiments, multiple doses are given over the course of days, weeks,months, or years. In some embodiments, a course of treatment is about 1or more doses (such as about 2 or more does, about 3 or more doses,about 4 or more doses, about 5 or more doses, about 7 or more doses,about 10 or more doses, about 15 or more doses, about 25 or more doses,about 40 or more doses, about 50 or more doses, or about 100 or moredoses).

In some embodiments, an administered dose of the pharmaceuticalcomposition is about 1 μg of protein per kg subject body mass or more(such as about 2 μg of protein per kg subject body mass or more, about 5μg of protein per kg subject body mass or more, about 10 μg of proteinper kg subject body mass or more, about 25 μg of protein per kg subjectbody mass or more, about 50 μg of protein per kg subject body mass ormore, about 100 μg of protein per kg subject body mass or more, about250 μg of protein per kg subject body mass or more, about 500 μg ofprotein per kg subject body mass or more, about 1 mg of protein per kgsubject body mass or more, about 2 mg of protein per kg subject bodymass or more, or about 5 mg of protein per kg subject body mass ormore).

In some embodiments, a therapeutic amount of a cell composition isadministered. Typically, precise amount of the compositions of thepresent invention to be administered can be determined by a physicianwith consideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising engineered cells, e.g. T cells, as described herein may beadministered at a dosage of 10⁴ to 10⁹ cells/kg body weight, such as 10⁵to 10⁶ cells/kg body weight, including all integer values within thoseranges. Engineered cell compositions, such as T cell compositions, mayalso be administered multiple times at these dosages. The cells can beadministered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al, New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regime for a particularpatient can readily be determined by one skilled in the art of medicineby monitoring the patient for signs of disease and adjusting thetreatment accordingly.

A variety of means are known for determining if administration of atherapeutic composition of the invention sufficiently modulatesimmunological activity by eliminating, sequestering, or inactivatingimmune cells mediating or capable of mediating an undesired immuneresponse; inducing, generating, or turning on immune cells that mediateor are capable of mediating a protective immune response; changing thephysical or functional properties of immune cells; or a combination ofthese effects. Examples of measurements of the modulation ofimmunological activity include, but are not limited to, examination ofthe presence or absence of immune cell populations (using flowcytometry, immunohistochemistry, histology, electron microscopy,polymerase chain reaction (PCR)); measurement of the functional capacityof immune cells including ability or resistance to proliferate or dividein response to a signal (such as using T-cell proliferation assays andpepscan analysis based on 3H-thymidine incorporation followingstimulation with anti-CD3 antibody, anti-T-cell receptor antibody,anti-CD28 antibody, calcium ionophores, PMA (phorbol 12-myristate13-acetate) antigen presenting cells loaded with a peptide or proteinantigen; B cell proliferation assays); measurement of the ability tokill or lyse other cells (such as cytotoxic T cell assays); measurementsof the cytokines, chemokines, cell surface molecules, antibodies andother products of the cells (e.g., by flow cytometry, enzyme-linkedimmunosorbent assays, Western blot analysis, protein microarrayanalysis, immunoprecipitation analysis); measurement of biochemicalmarkers of activation of immune cells or signaling pathways withinimmune cells (e.g., Western blot and immunoprecipitation analysis oftyrosine, serine or threonine phosphorylation, polypeptide cleavage, andformation or dissociation of protein complexes; protein array analysis;DNA transcriptional, profiling using DNA arrays or subtractivehybridization); measurements of cell death by apoptosis, necrosis, orother mechanisms (e.g., annexin V staining, TUNEL assays, gelelectrophoresis to measure DNA laddering, histology; fluorogenic caspaseassays, Western blot analysis of caspase substrates); measurement of thegenes, proteins, and other molecules produced by immune cells (e.g.,Northern blot analysis, polymerase chain reaction, DNA microarrays,protein microarrays, 2-dimensional gel electrophoresis, Western blotanalysis, enzyme linked immunosorbent assays, flow cytometry); andmeasurement of clinical symptoms or outcomes such as improvement ofautoimmune, neurodegenerative, and other diseases involvingself-proteins or self-polypeptides (clinical scores, requirements foruse of additional therapies, functional status, imaging studies) forexample, by measuring relapse rate or disease severity (using clinicalscores known to the ordinarily skilled artisan) in the case of multiplesclerosis, measuring blood glucose in the case of type I diabetes, orjoint inflammation in the case of rheumatoid arthritis.

VII. ARTICLES OF MANUFACTURE AND KITS

Also provided herein are articles of manufacture comprising thepharmaceutical compositions described herein in suitable packaging.Suitable packaging for compositions (such as ophthalmic compositions)described herein are known in the art, and include, for example, vials(such as sealed vials), vessels, ampules, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Thesearticles of manufacture may further be sterilized and/or sealed.

Further provided are kits comprising the pharmaceutical compositions (orarticles of manufacture) described herein, which may further compriseinstruction(s) on methods of using the composition, such as usesdescribed herein. The kits described herein may also include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for performing any methods described herein.

VIII. THERAPEUTIC APPLICATIONS

The pharmaceutical compositions described herein (includingpharmaceutical composition comprising the variant ICOSL polypeptides,the immunomodulatory proteins, the conjugates, the engineered cells andinfectious agents described herein) can be used in a variety oftherapeutic applications, such as the treatment of a disease. Forexample, in some embodiments the pharmaceutical composition is used totreat inflammatory or autoimmune disorders, cancer, organtransplantation, viral infections, and/or bacterial infections in amammal. The pharmaceutical composition can modulate (e.g. increase ordecrease) an immune response to treat the disease.

Such methods and uses include therapeutic methods and uses, for example,involving administration of the molecules or engineered cells, orcompositions containing the same, to a subject having a disease,condition, or disorder. In some cases, such as described, the disease ordisorder is an autoimmune or inflammatory disease or disorder. In somecases, such as described, the disease or disorder is a tumor or cancer.In some embodiments, the molecule or engineered cell is administered inan effective amount to effect treatment of the disease or disorder. Usesinclude uses of molecules containing a variant ICOSL polypeptide,immunomodulatory protein, conjugate, engineered cell and infectiousagents in such methods and treatments, and in the preparation of amedicament in order to carry out such therapeutic methods. In someembodiments, the methods are carried out by administering a variantICOSL polypeptide, immunomodulatory protein, conjugate, engineered cell,and infectious agent, or compositions comprising the same, to thesubject having or suspected of having the disease or condition. In someembodiments, the methods thereby treat the disease or condition ordisorder in the subject.

In some embodiments, the provided methods are applicable to therapeuticadministration of variant ICOSL polypeptides, the immunomodulatoryproteins, the conjugates, the engineered cells and infectious agentsdescribed herein. It is within the level of a skilled artisan, in viewof the provided disclosure, to choose a format for the indicationdepending on the type of modulation of the immune response, e.g.increase or decrease that is desired.

In some embodiments, a pharmaceutical composition provided herein thatstimulates the immune response is administered, which can be useful, forexample, in the treatment of cancer, viral infections, or bacterialinfections. In some embodiments, the pharmaceutical composition containsa variant ICOSL polypeptide in a format that exhibits agonist activityof its cognate binding partner CD28 or ICOS and/or that stimulates orinitiates costimulatory signaling via CD28 or ICOS. Exemplary formats ofan ICOSL polypeptide for use in connection with such therapeuticapplications include, for example, an immunomodulatory protein or“stack” of a variant ICOSL polypeptide and an IgSF domain or variantthereof that binds to a tumor antigen (e.g. Nkp30 or affinity-modifiedvariant) (also called a “tumor-localizing IgSF domain), a conjugatecontaining a variant ICOSL polypeptide linked to a tumor-targetingmoiety (also called a tumor-localizing moiety), an engineered cellexpressing a transmembrane immunomodulatory protein or an infectiousagent comprising a nucleic acid molecule encoding a transmembraneimmunomodulatory protein, such as for expression of the transmembraneimmunomodulatory protein in an infected cell (e.g. tumor cell or APC,e.g. dendritic cell).

Pharmaceutical compositions comprising engineered cells and the methodsdescribed herein can be used in adoptive cell transfer applications. Insome embodiments, cell compositions comprising engineered cells can beused in associated methods to, for example, modulate immunologicalactivity in an immunotherapy approach to the treatment of, for example,a mammalian cancer or, in other embodiments the treatment of autoimmunedisorders. The methods employed generally comprise a method ofcontacting a TIP of the present invention with a mammalian cell underconditions that are permissive to specific binding of the affinitymodified IgSF domain and modulation of the immunological activity of themammalian cell. In some embodiments, immune cells (such as tumorinfiltrating lymphocytes (TILs), T-cells (including CD8+ or CD4+T-cells), or APCs) are engineered to express as a membrane proteinand/or as a soluble variant ICOSL polypeptide, immunomodulatory protein,or conjugate as described herein. The engineered cells can then contacta mammalian cell, such as an APC, a second lymphocyte or tumor cell inwhich modulation of immunological activity is desired under conditionsthat are permissive of specific binding of the affinity modified IgSFdomain to a counter-structure on the mammalian cell such thatimmunological activity can be modulated in the mammalian cell. Cells canbe contacted in vivo or ex vivo.

In some embodiments, the engineered cells are autologous cells. In otherembodiments, the cells are allogeneic. In some embodiments, the cellsare autologous engineered cells reinfused into the mammal from which itwas isolated. In some embodiments, the cells are allogenic engineeredcells infused into the mammal. In some embodiments, the cells areharvested from a patient's blood or tumor, engineered to express apolypeptide (such as the variant ICOSL polypeptide, immunomodulatoryprotein, or conjugate as described herein), expanded in an in vitroculture system (for example, by stimulating the cells), and reinfusedinto the patient to mediate tumor destruction. In some embodiments, themethods are conducted by adoptive cell transfer wherein cells expressingthe TIP (e.g., a T-cell) are infused back into the patient. In someembodiments, the therapeutic compositions and methods of the inventionare used in the treatment of a mammalian patient of cancers such aslymphoma, lymphoid leukemia, myeloid leukemia, cervical cancer,neuroblastoma, or multiple myeloma.

In some embodiments, the pharmaceutical composition can be used toinhibit growth of mammalian cancer cells (such as human cancer cells). Amethod of treating cancer can include administering an effective amountof any of the pharmaceutical compositions described herein to a subjectwith cancer. The effective amount of the pharmaceutical composition canbe administered to inhibit, halt, or reverse progression of cancers.

The cancers that can be treated by the pharmaceutical compositions andthe treatment methods described herein include, but are not limited to,melanoma, bladder cancer, hematological malignancies (leukemia,lymphoma, myeloma), liver cancer, brain cancer, renal cancer, breastcancer, pancreatic cancer (adenocarcinoma), colorectal cancer, lungcancer (small cell lung cancer and non-small-cell lung cancer), spleencancer, cancer of the thymus or blood cells (i.e., leukemia), prostatecancer, testicular cancer, ovarian cancer, uterine cancer, gastriccarcinoma, a musculoskeletal cancer, a head and neck cancer, agastrointestinal cancer, a germ cell cancer, or an endocrine andneuroendocrine cancer. In some embodiments, the cancer is Ewing'ssarcoma. In some embodiments, the cancer is selected from melanoma, lungcancer, bladder cancer, and a hematological malignancy. In someembodiments, the cancer is a lymphoma, lymphoid leukemia, myeloidleukemia, cervical cancer, neuroblastoma, or multiple myeloma.

Human cancer cells can be treated in vivo, or ex vivo. In ex vivotreatment of a human patient, tissue or fluids containing cancer cellsare treated outside the body and then the tissue or fluids arereintroduced back into the patient. In some embodiments, the cancer istreated in a human patient in vivo by administration of the therapeuticcomposition into the patient.

In some embodiments, the pharmaceutical composition is administered as amonotherapy (i.e., as a single agent) or as a combination therapy (i.e.,in combination with one or more additional anticancer agents, such as achemotherapeutic drug, a cancer vaccine, or an immune checkpointinhibitor. In some embodiments, the pharmaceutical composition can alsobe administered with radiation therapy.

In some embodiments, the pharmaceutical composition is administered as amonotherapy (i.e., as a single agent) or as a combination therapy (i.e.,in combination with one or more additional anticancer agents, such as achemotherapeutic drug, a cancer vaccine, or an immune checkpointinhibitor. In some embodiments, the pharmaceutical composition can alsobe administered with radiation therapy. In some aspects, the immunecheckpoint inhibitor blocks PD-1 interactions with PD-L1 and/or PD-L2.In some cases, the immune checkpoint inhibitor is an antibody or antigenbinding fragment thereof that specifically binds PD-1, PD-L1 or PD-L2.In some cases, the immune checkpoint inhibitor is an anti-PD-1 antibody,such as nivolumab or pembrolizumab or an antigen binding fragmentthereof. In some cases, the immune checkpoint inhibitor blocks or is anantagonist of CTLA-4, such as is an anti-CTLA-4 antibody or antigenbinding fragment thereof. In some aspects of the present disclosure, theimmune checkpoint inhibitor is tremelimumab or ipilimumab.

In some embodiments, the pharmaceutical composition suppresses an immuneresponse, which can be useful in the treatment of inflammatory orautoimmune disorders, or organ transplantation. In some embodiments, thepharmaceutical composition contains a variant ICOSL polypeptide in aformat that exhibits antagonist activity of its cognate binding partnerCD28 or ICOS and/or that blocks or inhibits costimulatory signaling viaCD28 or ICOS. Exemplary formats of an ICOSL polypeptide for use inconnection with such therapeutic applications include, for example, avariant ICOSL polypeptide that is soluble (e.g. variant ICOSL-Fc fusionprotein), an immunomodulatory protein or “stack” of a variant ICOSLpolypeptide and another IgSF domain, including soluble forms thereofthat are Fc fusions, an engineered cell expressing a secretableimmunomodulatory protein, or an infectious agent comprising a nucleicacid molecule encoding a secretable immunomodulatory protein, such asfor expression and secretion of the secretable immunomodulatory proteinin an infected cell (e.g. tumor cell or APC, e.g. dendritic cell).

In some embodiments, the inflammatory or autoimmune disorder isantineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, avasculitis, an autoimmune skin disease, transplantation, a Rheumaticdisease, an inflammatory gastrointestinal disease, an inflammatory eyedisease, an inflammatory neurological disease, an inflammatory pulmonarydisease, an inflammatory endocrine disease, or an autoimmunehematological disease.

In some embodiments, the inflammatory and autoimmune disorders that canbe treated by the pharmaceutical composition described herein isAddison's Disease, allergies, alopecia areata, Alzheimer's,antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis,ankylosing spondylitis, antiphospholipid syndrome (Hughes Syndrome),asthma, atherosclerosis, rheumatoid arthritis, autoimmune hemolyticanemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmunelymphoproliferative syndrome, autoimmune myocarditis, autoimmuneoophoritis, autoimmune orchitis, azoospermia, Behcet's Disease, Berger'sDisease, bullous pemphigoid, cardiomyopathy, cardiovascular disease,celiac Sprue/coeliac disease, chronic fatigue immune dysfunctionsyndrome (CFIDS), chronic idiopathic polyneuritis, chronic inflammatorydemyelinating, polyradicalneuropathy (CIDP), chronic relapsingpolyneuropathy (Guillain-Barré syndrome), Churg-Strauss Syndrome (CSS),cicatricial pemphigoid, cold agglutinin disease (CAD), COPD (chronicobstructive pulmonary disease), CREST syndrome, Crohn's disease,dermatitis, herpetiformus, dermatomyositis, diabetes, discoid lupus,eczema, epidermolysis bullosa acquisita, essential mixedcryoglobulinemia, Evan's Syndrome, exopthalmos, fibromyalgia,Goodpasture's Syndrome, Graves' Disease, Hashimoto's thyroiditis,idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura(ITP), IgA nephropathy, immunoproliferative disease or disorder,inflammatory bowel disease (IBD), interstitial lung disease, juvenilearthritis, juvenile idiopathic arthritis (JIA), Kawasaki's Disease,Lambert-Eaton Myasthenic Syndrome, lichen planus, lupus nephritis,lymphocytic hypophysitis, Ménière's Disease, Miller Fish Syndrome/acutedisseminated encephalomyeloradiculopathy, mixed connective tissuedisease, multiple sclerosis (MS), muscular rheumatism, myalgicencephalomyelitis (ME), myasthenia gravis, ocular inflammation,pemphigus foliaceus, pemphigus vulgaris, pernicious anaemia,polyarteritis nodosa, polychondritis, polyglandular syndromes(Whitaker's syndrome), polymyalgia rheumatica, polymyositis, primaryagammaglobulinemia, primary biliary cirrhosis/autoimmune cholangiopathy,psoriasis, psoriatic arthritis, Raynaud's Phenomenon, Reiter'sSyndrome/reactive arthritis, restenosis, rheumatic fever, rheumaticdisease, sarcoidosis, Schmidt's syndrome, scleroderma, Sjörgen'sSyndrome, stiff-man syndrome, systemic lupus erythematosus (SLE),systemic scleroderma, Takayasu arteritis, temporal arteritis/giant cellarteritis, thyroiditis, Type 1 diabetes, ulcerative colitis, uveitis,vasculitis, vitiligo, interstitial bowel disease or Wegener'sGranulomatosis. In some embodiments, the inflammatory or autoimmunedisorder is selected from interstitial bowel disease, transplant,Crohn's disease, ulcerative colitis, multiple sclerosis, asthma,rheumatoid arthritis, and psoriasis.

In some embodiments, the inflammatory or autoimmune disorder is achronic autoimmune disease. In some embodiments, the inflammatory orautoimmune disorder is Sjogren's Syndrome (pSS) or Systemic LupusErythematosus (SLE). In some embodiments, the inflammatory or autoimmunedisorder is an inflammatory bowel disease (IBD). In some examples, theinflammatory or autoimmune disorder is Crohn's Disease. In someembodiments, the inflammatory or autoimmune disorder is an IBD-relateddisease or disorder, e.g. interstitial lung disease (ILD). In someembodiments, the inflammatory or autoimmune disorder is psoriaticarthritis or rheumatoid arthritis. In some embodiments, thepharmaceutical composition is administered to modulate an autoimmunecondition. For example, suppressing an immune response can be beneficialin methods for inhibiting rejection of a tissue, cell, or organtransplant from a donor by a recipient. Accordingly, in someembodiments, the pharmaceutical compositions described herein are usedto limit or prevent graft-related or transplant related diseases ordisorders, such as graft versus host disease (GvHD). In someembodiments, the pharmaceutical compositions are used to suppressautoimmune rejection of transplanted or grafted bone marrow, organs,skin, muscle, neurons, islets, or parenchymal cells.

In some embodiments, a pharmaceutical composition provided herein, suchas a variant ICOSL IgSF (e.g. IgV) Fc fusion protein provided herein, isused to treat psoriatic arthritis (PsA). In some cases, the PsA affectsone or more joints, such as fingers, toes, arms or legs, includingelbows, wrists, hands and feet, or sacroliliac joint. In some cases, thePsA is mild and/or affects four or less joints. In some cases the PsA ismoderate and/or affects four or more joints. In some cases, a subjectwith PsA may exhibit pain, stiffness or inflammation in the spine orneck, or in the one more joints.

In some embodiments, a pharmaceutical composition provided herein, suchas a variant ICOSL IgSF (e.g. IgV) Fc fusion protein provided herein, isused to treat rheumatoid arthritis (RA). In some cases, RA affectsjoints, lining of joints, and/or non-joint structures in the body (e.g.,skin, eyes, lungs, heart, kidneys, salivary glands, nerve tissue, bonemarrow or blood vessels). In some embodiments, RA or RA symptoms arechronic.

In some embodiments, a pharmaceutical composition provided herein, suchas a variant ICOSL IgSF (e.g. IgV) Fc fusion protein provided herein, isused to treat GVHD. In some embodiments, the GVHD is acute GVHD (aGVHD).In some cases, aGVHD occurs after allogeneic hematopoietic stem celltransplant (HSCT) and/or a reaction of donor immune cells against hosttissues. In some case, the aGVHD manifests in the skin, liver orgastrointestinal tract.

In some embodiments, a pharmaceutical composition provided herein, suchas a variant ICOSL IgSF (e.g. IgV) Fc fusion protein provided herein, isused to treat an autoimmune condition associated with an organtransplant. In some cases, treating the autoimmune condition associatedwith an organ transplant may prolong the survival of the host andtransplanted organ. In some embodiments, treating the autoimmunecondition associated with an organ transplant includes prophylaxis of orinhibiting or preventing transplant rejections by a subject that is therecipient of the organ transplant.

In some embodiments, a pharmaceutical composition provided herein, suchas a variant ICOSL IgSF (e.g. IgV) Fc fusion protein provided herein, isused to treat an inflammatory bowel disease (IBD). In some embodiments,a pharmaceutical composition provided herein, such as a variant ICOSLIgSF (e.g. IgV) Fc fusion protein provided herein, is used to treatCrohn's disease. In some embodiments, the Crohn's disease can include asubtype from Crohn's colitis, Crohn's enteritis, Crohn's iletis orCrohn's enterocolitis.

In some embodiments, a pharmaceutical composition provided herein, suchas a variant ICOSL IgSF (e.g. IgV) Fc fusion protein provided herein, isused to treat systemic lupus erythematosus (SLE). In some embodiments, apharmaceutical composition provided herein, such as a variant ICOSL IgSF(e.g. IgV) Fc fusion protein provided herein, is used to treat Sjogren'sSyndrome.

IX. EXEMPLARY EMBODIMENTS

Among the provided embodiments are:

1. A variant ICOS Ligand (ICOSL) polypeptide, comprising one or moreamino acid modifications in an immunoglobulin superfamily (IgSF) domainof an ICOSL reference polypeptide, wherein the ICOSL referencepolypeptide is a truncated extracellular domain comprising a contiguoussequence of amino acids comprising amino acids 1-112 and a C-terminaltruncation of at least 25 amino acids with reference to the ICOSLextracellular domain sequence set forth in SEQ ID NO: 32.

2. The variant ICOSL polypeptide of embodiment 1, wherein the variantICOSL polypeptide exhibits altered binding to the ectodomain(s) of ICOSor CD28 compared to the binding of the ICOSL reference polypeptide forthe same ectodomain(s).

3. The variant ICOSL polypeptide of embodiment 1 or embodiment 2,wherein the variant ICOSL polypeptide exhibits increased binding to theectodomain(s) of ICOS or CD28 compared to the binding of the ICOSLreference polypeptide for the same ectodomain(s).

4. The variant ICOSL polypeptide of any of embodiments 1-3, wherein theC-terminal truncation is of at least 30, at least 40, at least 50, atleast 60, at least 70, at least 80, at least 90, at least 100, at least125 amino acid residues.

5. The variant ICOSL polypeptide of any of embodiments 1-4, wherein theICOSL reference polypeptide is altered in or lacks a protease cleavagesite set forth as amino acids 204-209 of SEQ ID NO: 32.

6. The variant ICOSL polypeptide of any of embodiments 1-5, wherein theICOSL reference polypeptide comprises the sequence of amino acids setforth in SEQ ID NO: 545.

7. The variant ICOSL polypeptide of any of embodiments 1-5, wherein theICOSL reference polypeptide consists of the sequence of amino acids setforth in SEQ ID NO: 545.

8. A variant ICOSL Ligand (ICOSL) polypeptide, comprising one or moreamino acid modifications in an ICOSL reference polypeptide, wherein theICOSL reference polypeptide consists of the sequence of amino acids setforth in SEQ ID NO: 545.

9. A variant ICOS Ligand (ICOSL) polypeptide, comprising one or moreamino acid modifications in an immunoglobulin superfamily (IgSF) domainof an ICOSL reference polypeptide, wherein the ICOSL referencepolypeptide is altered in one or more amino acids corresponding to aminoacids 204-209 with reference to SEQ ID NO: 32.

10. The variant ICOSL polypeptide of embodiment 8 or embodiment 9,wherein the variant ICOSL polypeptide exhibits altered binding to one ormore of its binding partner(s) compared to the binding of the ICOSLreference polypeptide for the one or more binding partner(s).

11. The variant ICOSL polypeptide of embodiment 8 or embodiment 9,wherein the variant ICOSL polypeptide exhibits increased binding to oneor more of its binding partner(s) compared to the binding of the ICOSLreference polypeptide for the one or more binding partner(s).

12. The variant ICOSL polypeptide of any of embodiments 9-11, whereinthe alteration comprises a deletion of one or more contiguous aminoacids corresponding to amino acids 204-209 with reference to SEQ ID NO:32.

13. The variant ICOSL polypeptide of any of embodiments 1-6 and 9-12,wherein the ICOSL reference polypeptide comprises the sequence of aminoacids set forth in any of SEQ ID NOS: 600-605.

14. The variant ICOSL polypeptide of any of embodiments 1-6 and 9-12,wherein the ICOSL reference polypeptide consists of the sequence ofamino acids set forth in any of SEQ ID NOS: 600-605.

15. The variant ICOSL polypeptide of any of embodiments 1-6 and 9-14,wherein the alteration comprises at least one amino acid substitution atone or both of position 207 and 208 corresponding to positions set forthin SEQ ID NO: 32.

16. The variant ICOSL polypeptide of embodiment 15, wherein the at leastone amino acid substitution is N207A, N207G or L208G, or a conservativeamino acid substitution thereof.

17. The variant ICOSL polypeptide of any of embodiments 9-16, whereinthe reference ICOSL polypeptide comprises the sequence of amino acidsset forth in any of SEQ ID NOS: 623-628.

18. The variant ICOSL polypeptide of any of embodiments 9-17, whereinthe reference ICOSL polypeptide consists of the sequence of amino acidsset forth in any of SEQ ID NOS: 623-628.

19. The variant ICOSL polypeptide of any of embodiments 5-7 and 9-18,wherein the variant ICOSL polypeptide exhibits reduced proteolyticcleavage when expressed from a cell, optionally compared to afull-length extracellular domain of the variant ICOSL polypeptide whenexpressed from the same cell.

20. The variant ICOSL polypeptide of embodiment 19, wherein the cell isa mammalian cell.

21. The variant ICOSL polypeptide of embodiment 19 or embodiment 20,wherein the cell is a Chinese Hamster Ovary (CHO) cell line or aderivative thereof.

22. The variant ICOSL polypeptide of any of embodiments 1-21, whereinthe amino acid modification is an amino acid substitution, insertion ordeletion.

23. The variant of any of embodiments 1-22, wherein the one or moreamino acid modifications are in a position corresponding to position(s)selected from 10, 11, 13, 16, 18, 20, 25, 26, 27, 30, 33, 37, 38, 42,43, 47, 52, 54, 57, 61, 62, 67, 71, 72, 74, 75, 77, 78, 80, 84, 89, 90,92, 93, 94, 96, 97, 98, 99, 100, 102, 103, 107, 109, 110, 111, 113, 115,116, 117, 119, 120, 121, 122, 126, 129, 130, 132, 133, 135, 137, 138,139, 140, 142, 143, 144, 146, 151, 152, 153, 154, 155, 156, 158, 161,164, 166, 168, 172, 173, 175, 190, 192, 193, 194, 198, 201, 203, 207,208, 210, 212, 217, 218, 220, 221, 224, 225, or 227 with reference toSEQ ID NO:32.

24. The variant of any of embodiments 1-23, wherein the one or moreamino acid modifications are selected from M10V, M10I, V11E, S13G, E16V,S18R, A20T, A20V, S25G, R26S, F27C, F27S, N30D, Y33del, Q37R, T38P,K42E, T43A, Y47H, N52A, N52C, N52D, N52G, N52H, N52K, N52L, N52M, N52P,N52Q, N52R, N52S, N52T, N52V, N52Y, S54A, S54F, S54P, N57A, N57D, N57E,N57F, N57H, N57K, N57L, N57M, N57P, N57Q, N57S, N57T, N57V, N57W, N57Y,R61C, R61S, Y62F, L67P, A71T, G72R, L74Q, R75Q, D77G, F78L, L80P, N84Q,D89G, E90A, K92R, F93L, H94D, H94E, L96F, L96I, V97A, L98F, S99G, Q100A,Q100D, Q100E, Q100G, Q100K, Q100L, Q100M, Q100N, Q100P, Q100R, Q100S,Q100T, Q100V, L102R, G103E, V107A, V107I, S109G, S109N, V110A, V110D,V110N, E111del, T113E, H115Q, H115R, V116A, A117T, N119Q, F120I, F120S,S121G, V122A, V122M, S126R, S126T, H129P, S130G, S132F, Q133H, E135K,T137A, F138L, T139S, C140del, C140D, S142F, I143T, I143V, N144D, Y146C,V151A, Y152C, Y152H, W153R, I1154F, N155H, N155Q, K156M, D158G, L161M,L161P, Q164L, L166Q, N168Q, F172S, L173S, M175T, T190A, T190S, S192G,V193A, V193M, N194D, C198R, N201S, L203F, L203P, N207Q, L208P, V210A,S212G, D217G, D217V, I218N, I218T, E220G, R221G, R221I, R221K, I224V,T225A, T225S, N227K, or a conservative amino acid substitution thereof.

25. The variant ICOSL polypeptide of any of embodiments 1-24, whereinthe one or more amino acid modifications are in a position correspondingto position(s) 52, 57 or 100.

26. The variant ICOSL polypeptide of any of embodiments 1-25, whereinthe one or more amino acid modifications are selected from N52A, N52C,N52D, N52G, N52H, N52K, N52L, N52M, N52Q, N52R, N52S, N52T, N52V, N52Y,N52K, S54A, S54P, N57A, N57D, N57E, N57F, N57H, N57K, N57L, N57M, N57P,N57Q, N57S, N57T, N57V, N57Y, N57W, Q100A, Q100D, Q100G, Q100K, Q100L,Q100M, Q100N, Q100P, Q100R, Q100S, Q100T or Q100V.

27. The variant ICOSL polypeptide of any of embodiments 1-26, whereinthe one or more amino acid modifications are selected from amongN52Y/N57Y/F138L/L203P, N52H/N57Y/Q100P, N52S/Y146C/Y152C, N52H/C198R,N52H/C140D/T225A, N52H/C198R/T225A, N52H/K92R, N52H/S99G, N57Y/Q100P,N52S/S130G/Y152C, N52S/Y152C, N52S/C198R, N52Y/N57Y/Y152C,N52Y/N57Y/H129P/C198R, N52H/L161P/C198R, N52S/T113E, N52D/S54P,N52K/L208P, N52S/Y152H, N52D/V151A, N52H/I143T, N52S/L80P,N52S/R75Q/L203P, N52S/D158G, N52D/Q133H, N52S/N57Y/H94D/L96F/L98F/Q100R,N52S/N57Y/H94D/L96F/L98F/Q100R/G103E/F120S, N52S/G103E, N52H/F78L/Q100R,N52H/N57Y/Q100R/V110D, N52H/N57Y/R75Q/Q100R/V110D, N52H/N57Y/Q100R,N52H/N57Y/L74Q/Q100R/V10D, N52H/Q100R, N52H/S121G,A20V/N52H/N57Y/Q100R/S109G, N52H/N57Y/R61S/Q100R/V110D/L173S,N52H/N57Y/Q100R/V122A, N52H/N57Y/Q100R/F172S, N52H/N57Y, N52S/F120S,N52S/V97A, N52S/G72R, N52S/A71T/A117T, N52S/E220G,Y47H/N52S/V107A/F120S, N52H/N57Y/Q100R/V110D/S132F/M175T,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R,Q37R/N52H/N57Y/Q100R/V110N/S142F/C198R/D217V/R221G,N52H/N57Y/Q100R/V110D/C198R,N52H/N57Y/Q100R/V110D/V116A/L161M/F172S/S192G/C198R,F27S/N52H/N57Y/V110N, N52S/H94E/L96I/S109N/L166Q,S18R/N52S/F93L/I143V/R221G, A20T/N52D/Y146C/Q164L,V11E/N30D/N52H/N57Y/H94E/L96I/L98F/N194D/V210A/I218T,N52S/H94E/L96I/V122M, N52H/N57Y/H94E/L96I/F120I/S126T/W153R/I218N,M10V/S18R/N30D/N52S/S126R/T139S/L203F, S25G/N30D/N52S/F120S/N227K,N30D/N52S/L67P/Q100K/D217G/R221K/T225S,N52H/N57Y/Q100R/V110D/A117T/T190S/C198R,N52H/N57Y/Q100R/V110D/F172S/C198R,S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173S/C198R,N52H/N57Y/V110A/C198R/R221I,M1I/S13G/N52H/N57Y/D77G/V110A/H129P/I143V/F172S/V193M, C198R,N52H/N57Y/R61C/Y62F/Q100R/V110N/F120S/C198R,N52H/N57Y/Q100R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/N144D/F172S/C198R, N52S/H94E/L98F/Q100R,N52S/E90A, N30D/K42E/N52S, N52S/F120S/I143V/I224V,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/S54P, T38P/N52S/N57D,N52H/C140del/T225A, N52H/F78L/Q100R/C198R, N52H/N57Y/R75Q/Q100P/V110D,N52H/N57Y/L74Q/V110D/S192G, N52H/S121G/C198R, N52S/F120S/N227K,N52S/A71T/A117T/T190A/C198R, T43A/N52H/N57Y/L74Q/D89G/V110D/F172S,N52H/N57Y/Q100R/V110D/S132F/M175T, N52D,N52H/N57Y/Q100R/V107I/V110D/I154F/C198R/R221G, N52Q/N207Q, N168Q/N207Q,N52Q/N168Q, N52Q/N84Q, N52Q/N119Q, N52Q/N84Q/N168Q, N52Q/N84Q/N207Q,N52Q/N119Q/N155Q, N52H/N84Q/N119Q, N52H/N84Q, N52H/N84Q/N168Q/N207Q,N52Q/N84Q/N155Q/N168Q, N52Q/N84Q/N119Q/N168Q, N52Q/N84Q/N119Q/N207Q,N52Q/N84Q/N119Q/N155Q, N52Q/N84Q/N119Q/N155Q/N207Q, N52Y/F138L/L203P,N57Y/Q100R/C198R, N57Y/F138L/L203P, Q100R/F138L,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R,N52H/N57Y/Q100R/L102R/H115R/F172S/C198R, N52H/V122A/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/N194D, N52H/N57Y/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/H115R, N52H/N57Y/Q100R/H115R,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/F172S, N52H/Q100R/H115R/I143T/F172S,N52H/N57Y/Q100P/H115R/F172S, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/E90A/H115R, N30D/K42E N52S/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N52S/H115R/F120S/I143V/C198R, N52S/H115R/F172S/C198R,N52H/N57Y/Q100P/C198R, N52H/N57Y/Q100P H115R/F172S/C198R,N52H/N57Y/Q100P/F172S/C198R, N52H/N57Y/Q100P/H115R,N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R, N52H/Q100R/H115R/F172S,N52H/Q100R/F172S/C198R, N52H/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/F172S/C198R, N52A/N57F/Q100S, N52A/N57H/Q100S,N52A/N57Y/Q100A, N52D/N57A/Q100A, N52D/Q100S, N52G/Q100A, N52H/Q100A,N52M/N57H/Q100S, N52M/N57W/Q100P, N52Q/N57F, N52Q/N57S/Q100A,N52R/N57L/Q100A, N52R/N57Y/Q100P, N52R/N57Y/Q100S, N52S/N57A/Q100A,N52S/N57H/Q100E, N52S/N57L/Q100S, N52S/N57M/Q100S, N52S/N57Y/Q100S,N52S/N57Y/Q100M, N52S/N57Y/Q100V, N52T/N57H/Q100S, N52T/N57H/Q100A,N52T/N57Y/Q100A, N52V/N57L/Q100A, N52H/N57Y/Q100K, N52K/N57Y/Q100R,N52L/N57H/Q100R, N52R/N57F/Q100N, N52R/N57F/Q100P, N52R/N57F/Q100R,N52R/N57F/Q100T, N52R/N57H/Q100K, N52R/N57L/Q100S, N52R/N57W/Q100K,N52R/N57W, N52R/N57Y/Q100R, N52C/N57E/Q100S, N52G/N57P/Q100D,N52G/N57V/Q100G, N52G/N57V, N52L/N57V, N52P/N57P, N52P/N57S/Q100G,N52S/N57L/Q100G, N52T/N57K/Q100P, N52V/N57T/Q100L, N57Q/Q100P, orR26S/N52H/N57Y/V110D/T137A/C198R.

28. The variant ICOSL polypeptide of any of embodiments 1-24, whereinthe one or more amino acid modifications are selected from amongF120S/Y152H/N201S, E111del, Y33del, N168Q/N207Q, N84Q/N207Q,N155Q/N207Q, N119Q/N168Q, N119Q/N207Q, N119Q/N155Q, N84Q/N119Q,N84Q/N155Q/N168Q, N84Q/N168Q/N207Q, N84Q/N155H/N207Q, N155Q/N168Q/N207Q,N119Q N155Q/N168Q, N119Q/N168Q/N207Q, N84Q/N119Q/N207Q,N119Q/N155H/N207Q, N84Q/N119Q/N155Q, N84Q/N119Q/N155Q/N168Q,N84Q/N155Q/N168Q/N207Q, N84Q/N119Q/N155Q/N207Q,N84Q/N119Q/N155Q/N168Q/N207Q or F138L/L203P.

29. The variant ICOSL polypeptide of any of embodiments 1-28, whereinthe one or more amino acid modifications are selected from C198R, D158G,E16V, E90A, F120S, F138L, F172S, H115R, H115X, I143T, I143V, I224V,K156M, K42E, K92R, L102R, L203P, L208P, N194D, N30D, N52A, N52D, N52G,N52H, N52K, N52L, N52M, N52Q, N52R, N52S, N52T, N52Y, N57F, N57H, N57K,N57L, N57M, N57P, N57S, N57V, N57W, N57Y, Q100A, Q100D, Q100E, Q100K,Q100M, Q100P, Q100P, Q100R, Q100S, Q100T, Q133H, R221I, R75Q, S54A,S54P, T113E, T225A, V110D, V122A, Y146C, or Y152C; or

A117T, A20V, A71T, A91G, A91G, AE88D, C140del, C198R, D158G, D77G, D90K,E117G, E135K, E16V, E81A, E88D, E90A, F120I, F120S, F138L, F172S, F27C,F92Y, G72R, H115R, H115X, H129P, H94E, I118V, I127T, I143T, I143V,I154F, I218N, I218T, I224V, K156M, K169E, K36G, K42E, K89R, K92R, K93R,L102R, L161P, L166Q, L173S, L203F, L203P, L208P, L209P, L40M, L70Q,L70R, L74Q, L80P, L96I, L98F, M10I, M10V, N115Q, N119Q, N122S, N144D,N155X, N168Q, N168X, N178S, N194D, N207Q, N207X, N227K, N25S, N30D,N52A, N52D, N52G, N52H, N52K, N52L, N52M, N52Q, N52R, N52S, N52T, N52V,N52Y, N57A, N57F, N57H, N57L, N57M, N57S, N57V, N57W, N57Y, N63S, N84Q,Q100A, Q100E, Q100G, Q100K, Q100M, Q100N, Q100P, Q100R, Q100S, Q100T,Q100V, Q133H, R221G, R221I, S109G, S109N, S114T, S121G, S126R, S126T,S130G, S132F, S13G, S18R, S192G, S212G, S25G, S54A, S54P, S99G, T113E,T120S, T130A, T139S, T190A, T199S, T225A, T41I, V107I, V110A, V110D,V11E, V122A, V122M, V193M, V210A, W153R, Y146C, Y152C, or Y152H.

30. The variant ICOSL polypeptide of any of embodiments 1-29, whereinthe one or more amino acid modifications are selected from among N52S,N52H, N52D, N52H/N57Y/Q100P, N52S/Y146C/Y152C, N52H/C198R,N52H/C198R/T225A, N52H/K92R, N57Y, N52S/C198R, N52S/T113E, S54A,N52D/S54P, N52K/L208P, N52H/I143T, N52S/R75Q/L203P, N52S/D158G,N52D/Q133H, N52H/N57Y/Q100R/V122A, N52H/N57Y/Q100R/F172S,N52H/N57Y/Q100R, N52S/N194D, N52H/N57Y/Q100R/L102R/V110D/H115R/C198R,N52S/E90A, N52S/F120S/I143V/I224V, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/H115R/F120S/I143V/C198R, N52H/N57Y/Q100P/C198R,N52H/N57Y/Q100P/H115R/F172S/C198R, N52H/N57Y/Q100P/F172S/C198R,N52H/N57Y/Q100P/H115R, N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R,N52H/Q100R/H115X/F172S/C198R, N52H/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/H115R/F172S/C198R, Q100R, N52Y/F138L/L203P,N57Y/Q100R/C198R, N57Y/F138L/L203P, N57Y/Q100P, Q100R/F138L,N52H/N57Y/Q100R/H115R, N52H/N57Y/Q100R/F172S,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R, N52H/N57Y/Q100R/L102R,H115R/F172S/C198R, N52H/N57Y/Q100R/H115R F172S/N194D,N52H/N57Y/H115R/F172S/C198R, N52H/N57Y/Q100R/H115R/C198R,N52H/N57Y/H115R, N52H/Q100R/H115R/I143T F172S,N52H/N57Y/Q100P/H115R/F172S, E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,N52S/E90A/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N30D/K42E/N52S/H115R, N52S/E90A/H115R, N30D/K42E/N52S/H115R,N52A/N57H/Q100S, N52A/N57Y/Q100A, N52D/Q100S, N52G/Q100A,N52M/N57H/Q100S, N52M/N57W/Q100P, N52Q/N57S/Q100A, N52R/N57L/Q100A,N52S/N57H/Q100E, N52S/N57L/Q100S, N52S/N57M/Q100S, N52S/N57Y/Q100M,N52T/N57H/Q100S, N52R/N57F/Q100P, N52R/N57F/Q100T, N52R/N57W/Q100K,N52R/N57W, N52G/N57P/Q100D, N52G/N57V/Q100G, N52G/N57V, N52L/N57V,N52S/N57L/Q100G or N52T/N57K/Q100P; or

N52S, N52H, N52D, N52Y/N57Y/F138L/L203P, N52H/N57Y/Q100P,N52S/Y146C/Y152C, N52H/C198R, N52H/C140del/T225A, N52H/C198R/T225A,N52H/K92R, N52H/S99G, N57Y, N57Y/Q100P, N52S/S130G/Y152C, N52S/Y152C,N52S/C198R, N52Y/N57Y/Y152C, N52Y/N57Y/H129P/C198R, N52H/L161P/C198R,N52S/T113E, S54A, N52D/S54P, N52K/L208P, N52S/Y152H, N52H/I143T,N52S/L80P, N52S/D158G, N52D/Q133H, L70Q/A91G/N144D,L70Q/A91G/E117G/I118V/T120S/T130A, L70R/A91G/I118V/T120S/T130A/T199S,L70Q/E81A/A91G/I118V/T120S/I127T/T130A,N63S/L70Q/A91G/S114T/I118V/T120S/T130A, T41I/A91G,E88D/K89R/D90K/A91G/F92Y/K93R/N122S/N178S,E88D/K89R/D90K/A91G/F92Y/K93R, AE88D/K89R/D90K/A91G/F92Y/K93R,K36G/L40M, N52H/N57Y/Q100R/V122A, N52H/N57Y/Q100R/F172S,N52H/N57Y/Q100R, N52S/F120S/N227K, N52S/N194D, N52S/F120S, N52S/G72R,N52S/A71T/A117T/T190A/C198R,N52H/N57Y/Q100R/V107I/V110D/S132F/I154F/C198R/R221G,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R,N52H/N57Y/Q100R/V110D/C198R,V11E/N30D/N52H/N57Y/H94E/L96I/L98F/N194D/V210A/I218T,N52S/H94E/L96I/V122M, N52H/N57Y/H94E/L96I/F120I/S126T/W153R/I218N,M10V/S18R/N30D/N52S/S126R/T139S/L203F, S25G/N30D/N52S/F120S/N227K,N52H/N57Y/Q100R/V110D/F172S/C198R,S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173S/C198R,N52H/N57Y/V110A/C198R/R221I,M10I/S13G/N52H/N57Y/D77G/V110A/H129P/I143V/F172S/V193M, C198R,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/N144D/F172S/C198R, N52S/H94E/L98F/Q100R,N52S/E90A, N52S/F120S/I143V/I224V, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/H115R/F120S/I143V/C198R, N52H/N57Y/Q100P/C198R,N52H/N57Y/Q100P/H115R/F172S/C198R, N52H/N57Y/Q100P/F172S/C198R,N52H/N57Y/Q100P/H115R, N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R,N52H/Q100R/H115R/F172S, N52H/Q100R/H115X/F172S/C198R,N52H/Q100R/H115R/F172S/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/H115R/F172S/C198R, N52S/H94E/L96I/S109N/L166Q/,N52H/N57Y/Q100R/C198R, N52H/N57Y/L74Q/V110D/S192G, N52H/Q100R,N52H/S121G/C198R, A20V/N52H/N57Y/Q100R/S109G, N52H/N57Y/Q100P/C198R,N52H/N57Y/Q100R/V110D/C198R/S212G, L70Q/A91G/I118A/T120S/T130A/K169E,Q100R, N52Y/F138L/L203P, N57Y/Q100R/C198R, N57Y/F138L/L203P, N52H, N57Y,N57Y/Q100P, N52H/N57Y/Q100R/H115R, N52H/N57Y/Q100R/F172S,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R, N52H/N57Y/Q100R/L102RH115R/F172S/C198R, N52H/N57Y/Q100R/H115R F172S/N194D,N52H/N57Y/H115R/F172S/C198R, N52H/N57Y/Q100R/H115R/C198R,N52H/N57Y/H115R, N52H/Q100R/H115R/I143T F172S,N52H/N57Y/Q100P/H115R/F172S, E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,N30D/K42E/N52S/H115R/C198R R221I, N52S/E90A/H115R, N30D/K42E/N52S/H115R,N52S/H115R/F172S/C198R, N119Q, N207Q, N52Q/N207X, N168X/N207X,N52Q/N168Q, N84Q/N207Q, N119Q N155X, N52Q/N119Q, N52Q/N84Q/N207Q,N119Q/N155Q/N168Q, N52H/N84Q/N119Q, N52Q/N84Q/N155X/N168X,N52A/N57F/Q100S, N52A/N57H/Q100S, N52A/N57Y/Q100A, N52D/N57A/Q100A,N52D/Q100S, N52G/Q100A, N52H/Q100A, N52M/N57H/Q100S, N52M/N57W/Q100P,N52Q/N57F, N52Q/N57S/Q100A, N52R/N57L/Q100A, N52R/N57Y/Q100P,N52R/N57Y/Q100S, N52S/N57A/Q100A, N52S/N57H/Q100E, N52S/N57L/Q100S,N52S/N57M/Q100S, N52S/N57Y/Q100S, N52S/N57Y/Q100M, N52S/N57Y/Q100V,N52T/N57H/Q100S, N52T/N57H/Q100A, N52T/N57Y/Q100A, N52V/N57L/Q100A,N52H/N57Y/Q100K, N52K/N57Y/Q100R, N52L/N57H/Q100R, N52R/N57F/Q100N,N52R/N57F/Q100P, N52R/N57F/Q100R, N52R/N57F/Q100T, N52R/N57L/Q100S,N52R/N57W/Q100K, N52R/N57W, N52G/N57V, N52L/N57V, N52S/N57L/Q100G, orN52T/N57K/Q100P.

31. The variant ICOSL polypeptide of any of embodiments 1-30, whereinthe variant ICOSL polypeptide exhibits increased binding to theectodomain of ICOS or CD28 compared to the binding of the referenceICOSL polypeptide to the same ectodomain.

32. The variant ICOSL polypeptide of any of embodiments 1-31, whereinthe one or more amino acid modifications are selected from C198R, D158G,E16V, E90A, F120S, F138L, F172S, H115R, I143V, I224V, K156M, K42E, K92R,L102R, L203P, L208P, N194D, N30D, N52A, N52D, N52G, N52H, N52K, N52L,N52M, N52Q, N52R, N52S, N52T, N52Y, N57F, N57H, N57L, N57M, N57S, N57V,N57W, N57Y, Q100A, Q100E, Q100G, Q100K, Q100M, Q100P, Q100R, Q100S,Q133H, S212G, S54A, S54P, T113E, V110D, V122A, Y146C, Y152C, or T225A.

33. The variant ICOSL polypeptide of any of embodiments 1-33, whereinthe one or more amino acid modifications are selected from amongN52A/N57Y/Q100A, N52D/Q100S, N52G/Q100A, N52M/N57H/Q100S,N52M/N57W/Q100P, N52Q/N57S/Q100A, N52R/N57L/Q100A, N52S/N57H/Q100E,N52S/N57L/Q100S, N52S/N57M/Q100S, N52S/N57Y/Q100M, N52T/N57H/Q100S,N52R/N57F/Q100P, N52R/N57F/Q100T, N52R/N57W/Q100K, N52R/N57W, N52G/N57V,N52L/N57V, N52S/N57L/Q100G, N52T/N57K/Q100P, N52S, N52H, N52D,N52Y/N57Y/F138L/L203P, N52H/N57Y/Q100P, N52S/Y146C/Y152C, N52H/C198R,N52H/C198R/T225A, N52H/K92R, N57Y, N52S/C198R, N52S/T113E, S54A,N52D/S54P, N52K/L208P, N52H/I143T, N52S/D158G, N52D/Q133H,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/V122A,N52H/N57Y/Q100R/F172S, N52H/N57Y/Q100R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/E90A,N52S/F120S/I143V/I224V, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/H115R/F120S/I143V/C198R, N52H/N57Y/Q100P/C198R,N52H/N57Y/Q100P/H115R/F172S/C198R, N52H/N57Y/Q100P/F172S/C198R,N52H/N57Y/Q100P/H115R, N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R,N52H/Q100R/H115R/F172S, N52H/Q100R/H115X/F172S/C198R,N52H/Q100R/H115R/F172S/C198R, N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S, N52H/N57Y/Q100R/H115R/F172S/C198R, Q100R,N52Y/F138L/L203P, N57Y/Q100R/C198R, N57Y/F138L/L203P, N52H, N57Y,N57Y/Q100P, Q100R/F138L, N52H/N57Y/Q100R/H115R, N52H/N57Y/Q100R/F172S,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R, N52H/N57Y/Q100R/L102RH115R/F172S/C198R, N52H/N57Y/Q100R/H115R F172S/N194D,N52H/N57Y/H115R/F172S/C198R, N52H/N57Y/Q100R/H115R/C198R,N52H/N57Y/H115R, N52H/Q100R/H115R/I143T/F172S,N52H/N57Y/Q100P/H115R/F172S, E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,N52S/E90A/H115R, N52S/E90A/H115R, or N30D/K42E/N52S/H115R.

34. The variant ICOSL polypeptide of any of embodiments 1-33, whereinthe variant ICOSL polypeptide exhibits increased binding to theectodomain of ICOS and CD28 compared to the binding of the referenceICOSL polypeptide to the same ectodomains.

35. The variant ICOSL polypeptide of any of embodiments 1-34, whereinthe variant ICOSL polypeptide comprises the sequence of amino acids setforth in any one of SEQ ID NOS: 546-599, 734-781, 783, 786, 788, 792,796, 798, 800, 802, 804, 806, 808, 811, 813, 815, 817, 818, 820, 822,824, 826, 827, 829, 831, 833, 834, 836, 838, 840-843, 845, 847, 848,850-853, 855, 857, 907, 910, or a sequence of amino acids that exhibitsat least 90%, 91%%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to any one of SEQ ID NOS: 546-599, 734-781, 783, 786, 788, 792,796, 798, 800, 802, 804, 806, 808, 811, 813, 815, 817, 818, 820, 822,824, 826, 827, 829, 831, 833, 834, 836, 838, 840-843, 845, 847, 848,850-853, 855, 857, 907, 910.

36. The variant ICOSL polypeptide of any of embodiments 1-34, whereinthe variant ICOSL polypeptide consists of the sequence of amino acidsset forth in any one of SEQ ID NOS: 546-599, 734-781, 783, 786, 788,792, 796, 798, 800, 802, 804, 806, 808, 811, 813, 815, 817, 818, 820,822, 824, 826, 827, 829, 831, 833, 834, 836, 838, 840-843, 845, 847,848, 850-853, 855, 857, 907, 910, or a sequence of amino acids thatexhibits at least 90%, 91%%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to any one of SEQ ID NOS: 546-599, 734-781, 783, 786,788, 792, 796, 798, 800, 802, 804, 806, 808, 811, 813, 815, 817, 818,820, 822, 824, 826, 827, 829, 831, 833, 834, 836, 838, 840-843, 845,847, 848, 850-853, 855, 857, 907, or 910.

37. A variant ICOS Ligand (ICOSL) polypeptide, comprising an IgV domainor specific binding fragment thereof, an IgC domain or a specificbinding fragment thereof, or both, wherein the variant ICOSL polypeptidecomprises one or more amino acid modifications in an ICOSL referencepolypeptide or a specific binding fragment thereof corresponding toamino acid modifications are selected from N52A, N52C, N52D, N52G, N52K,N52L, N52M, N52R, N52T, N52V, N57A, N57E, N57F, N57H, N57K, N57L, N57M,N57P, N57Q, N57S, N57T, N57V, N57W, Q100A, Q100D, Q100G, Q100L, Q100M,Q100N, Q100R, Q100S, Q100T or Q100V. with reference to SEQ ID NO:32.

38. The variant ICOSL polypeptide of embodiment 37, wherein the one ormore amino acid modifications are selected from among N52A/N57F/Q100S,N52A, /N57H/Q100S, N52A/N57Y/Q100A, N52D/N57A/Q100A, N52D/Q100S,N52G/Q100A, N52H/Q100A, N52M/N57H/Q100S, N52M/N57W/Q100P, N52Q/N57F,N52Q/N57S/Q100A, N52R/N57L/Q100A, N52R/N57Y/Q100P, N52R/N57Y/Q100S,N52S/N57A/Q100A, N52S/N57H/Q100E, N52S/N57L/Q100S, N52S/N57M/Q100S,N52S/N57Y/Q100S, N52S/N57Y/Q100M, N52S/N57Y/Q100V, N52T/N57H/Q100S,N52T/N57H/Q100A, N52T/N57Y/Q100A, N52V/N57L/Q100A, N52H/N57Y/Q100K,N52K/N57Y/Q100R, N52L/N57H/Q100R, N52R/N57F/Q100N, N52R/N57F/Q100P,N52R/N57F/Q100R, N52R/N57F/Q100T, N52R/N57H/Q100K, N52R/N57L/Q100S,N52R/N57W/Q100K, N52R/N57W, N52R/N57Y/Q100R, N52C/N57E/Q100S,N52G/N57P/Q100D, N52G/N57V/Q100G, N52G/N57V, N52L/N57V, N52P/N57P,N52P/N57S/Q100G, N52S/N57L/Q100G, N52T/N57K/Q100P, N52V/N57T/Q100L orN57Q/Q100P.

39. The variant ICOSL polypeptide of embodiment 37 or embodiment 38,wherein the ICOSL reference polypeptide is a mammalian ICOSL or aspecific binding fragment thereof.

40. The variant ICOSL polypeptide of any of embodiments 37-39, whereinthe ICOSL reference polypeptide is a human ICOSL or a specific bindingfragment thereof.

41. The variant ICOSL polypeptide of any one of embodiments 37-40,wherein the ICOSL reference polypeptide comprises (i) the sequence ofamino acids set forth in SEQ ID NO:32, (ii) a sequence of amino acidsthat has at least 95% sequence identity to SEQ ID NO:32; or (iii) aportion of (i) or (ii) comprising an IgV domain or IgC domain orspecific binding fragments thereof or both.

42. The variant ICOSL polypeptide of any one of embodiments 37-41,wherein:

the specific binding fragment of the IgV domain or IgC domain has alength of at least 50, 60, 70, 80, 90, 100, 110 or more amino acids; or

the specific binding fragment of the IgV domain comprises a length thatis at least 80% of the length of the IgV domain set for as amino acids19-129 of SEQ ID NO:5 and/or the specific binding fragment of the IgCdomain comprises a length that is at least 80% of the length of the IgCdomain set forth as amino acids 141-227 of SEQ ID NO:5.

43. The variant ICOSL polypeptide of any of embodiments 37-42, whereinthe variant ICOSL polypeptide comprises the IgV domain or a specificfragment thereof and the IgC domain or a specific fragment thereof.

44. The variant ICOSL polypeptide of any of embodiments 37-43, whereinthe variant ICOSL polypeptide comprises the sequence of amino acids setforth in any one of SEQ ID NOS: 638-685, 905, 908, or a sequence ofamino acids that exhibits at least 90%, 91%%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity to any one of SEQ ID NOS: 638-685,905, 908.

45. The variant ICOSL polypeptide of any of embodiments 37-43, whereinthe variant ICOSL polypeptide consists of the sequence of amino acidsset forth in any one of SEQ ID NOS: 638-685, 905, 908, or a sequence ofamino acids that exhibits at least 90%, 91%%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity to any one of SEQ ID NOS: 638-685,905, 908.

46. The variant ICOSL polypeptide of any of embodiments 37-43, whereinthe variant ICOSL polypeptide comprises the IgV domain or a specificbinding fragment thereof.

47. The variant ICOSL polypeptide of any of embodiments 37-43 and 46,wherein the variant ICOSL polypeptide comprises the sequence of aminoacids set forth in any one of SEQ ID NOS: 686-781, 907, 910, or asequence of amino acids that exhibits at least 90%, 91%%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOS:686-781, 907, 910.

48. The variant ICOSL polypeptide of any of embodiments 37-43 and 46,wherein the variant ICOSL polypeptide consists of the sequence of aminoacids set forth in any one of SEQ ID NOS: 686-781, 907, 910, or asequence of amino acids that exhibits at least 90%, 91%%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOS:686-781, 907, 910.

49. The variant ICOSL polypeptide of any of embodiments 37-43 and 46-48,wherein the IgV domain or specific binding fragment thereof is the onlyICOSL portion of the variant ICOSL polypeptide.

50. The variant ICOSL polypeptide of any of embodiments 37-42, whereinthe IgC domain or specific binding fragment thereof is the only ICOSLportion of the variant ICOSL polypeptide.

51. The variant ICOSL polypeptide of any of embodiments 37-50, whereinthe variant ICOSL polypeptide exhibits altered binding to the ectodomainof ICOS or CD28 compared to the binding of the ICOSL referencepolypeptide for the same ectodomain.

52. The variant ICOSL polypeptide of any of embodiments 37-51, whereinthe variant ICOSL polypeptide exhibits increased binding to theectodomain(s) of ICOS or CD28 compared to the binding of the ICOSLreference polypeptide for the same ectodomain(s).

53. The variant ICOSL polypeptide of any of embodiments 1-52, whereinthe binding is increased more than 1.2-fold, 1.5-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold,30-fold, 40-fold, 50-fold or 60-fold.

54. The variant ICOSL polypeptide of any of embodiments 1-53, whereinthe ICOS is a human ICOS.

55. The variant ICOSL polypeptide of any of embodiments 1-54, whereinthe CD28 is a human CD28.

56. The variant ICOSL polypeptide of any of embodiments 1-55, whereinthe variant ICOSL polypeptide exhibits decreased binding to theectodomain of CTLA-4 compared to the binding of the reference ICOSLpolypeptide for the same ectodomain.

57. The variant ICOSL polypeptide of embodiment 56, wherein the bindingis decreased more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold,40-fold, 50-fold or 60-fold.

58. The variant ICOSL polypeptide of any of embodiments 1-57, whereinthe CTLA-4 is a human CTLA-4.

59. The variant ICOSL polypeptide of any of embodiments 1-58, whereinthe altered (increased or decreased) binding is altered (increased ordecreased) binding affinity.

60. The variant ICOSL polypeptide of any of embodiments 1-59, whereinthe variant ICOSL polypeptide comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications,optionally amino acid substitutions, insertions and/or deletions.

61. The variant ICOSL polypeptide of any of embodiments 1-60, whereinthe variant ICOSL polypeptide exhibits at least or at least about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to theICOSL reference polypeptide.

62. The variant ICOSL polypeptide of any of embodiments 1-61 that is asoluble protein.

63. The variant ICOSL polypeptide of any of embodiments 1-62, wherein:

the variant ICOSL polypeptide lacks a transmembrane domain andintracellular signaling domain; and/or

when expressed from a cell, the variant ICOSL polypeptide is notexpressed on the surface of the cell.

64. The variant ICOSL polypeptide of any of embodiments 1-61, whereinthe variant ICOSL polypeptide further comprises a transmembrane domain.

65. The variant ICOSL polypeptide of embodiment 64, wherein thetransmembrane domain comprises the sequence of amino acids set forth asresidues 257-277 of SEQ ID NO:5 or a functional variant thereof thatexhibits at least 85% sequence identity to residues 257-277 of SEQ IDNO:5.

66. The variant ICOSL polypeptide of embodiment 64 or embodiment 65,further comprising a cytoplasmic signaling domain linked to thetransmembrane domain.

67. The variant ICOSL polypeptide of embodiment 66, wherein thecytoplasmic signaling domain comprises the sequence of amino acids setforth as residues 278-302 of SEQ ID NO:5 or a functional variant thereofthat exhibits at least 85% sequence identity to residues 278-302 of SEQID NO:5.

68. The variant ICOSL polypeptide of any of embodiments 1-67 that isdeglycosylated or partially deglycosylated compared to the ICOSLreference sequence.

69. An immunomodulatory protein, comprising the variant ICOSLpolypeptide of any of embodiments 1-68 and a half-life extending moiety.

70. The immunomodulatory protein of embodiment 69, wherein the half-lifeextending moiety comprises a multimerization domain, albumin, analbumin-binding polypeptide, Pro/Ala/Ser (PAS), a C-terminal peptide(CTP) of the beta subunit of human chorionic gonadotropin, polyethyleneglycol (PEG), long unstructured hydrophilic sequences of amino acids(XTEN), hydroxyethyl starch (HES), an albumin-binding small molecule, ora combination thereof.

71. The immunomodulatory protein of embodiment 69 or embodiment 70,wherein the half-life extending moiety is or comprises Pro/Ala/Ser (PAS)and the variant ICOSL polypeptide is PASylated.

72. The immunomodulatory protein of embodiment 71, wherein the half-lifeextending moiety comprises the sequence set forth in SEQ ID NO: 904.

73. The immunomodulatory protein of embodiment 69 or embodiment 70,wherein the half-life extending moiety is or comprises a multimerizationdomain.

74. The immunomodulatory protein of embodiment 73, wherein themultimerization domain is selected from an Fc region of animmunoglobulin, a leucine zipper, an isoleucine zipper or a zinc finger.

75. The immunomodulatory protein of embodiment 73 or embodiment 74,wherein the variant ICOSL polypeptide is linked, directly or indirectlyvia a linker, to the multimerization domain.

76. The immunomodulatory protein of any of embodiments 73-75, whereinthe immunomodulatory protein is a multimer comprising a first variantICOSL polypeptide linked to a first multimerization domain and a secondvariant ICOSL polypeptide linked to a second multimerization domain,wherein the first and second multimerization domains interact to form amultimer comprising the first and second variant ICOSL polypeptide.

77. The immunomodulatory protein of embodiment 76, wherein the multimeris a dimer.

78. The immunomodulatory protein of embodiment 76 or embodiment 77,wherein the first variant ICOSL polypeptide and the second variant ICOSLpolypeptide are the same.

79. The immunomodulatory protein of embodiment 77 or embodiment 78,wherein the dimer is a homodimer.

80. The immunomodulatory protein of embodiment 77, wherein the dimer isa heterodimer.

81. The immunomodulatory protein of any of embodiments 73-80, whereinthe multimerization domain is or comprises an Fc region of animmunoglobulin.

82. The immunomodulatory protein of embodiment 81, wherein the Fc regionis of an immunoglobulin G1 (IgG1) or an immunoglobulin G2 (IgG2)protein.

83. The immunomodulatory protein of embodiment 81 or embodiment 82,wherein the immunoglobulin protein is human and/or the Fc region ishuman.

84. The immunomodulatory protein of any of embodiments 81-83, whereinthe Fc region comprises the sequence of amino acids set forth in SEQ IDNO: 227 or a variant thereof that exhibits at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:227.

85. The immunomodulatory protein of any of embodiments 81-84, whereinthe Fc region comprises the sequence of amino acids set forth in SEQ IDNO: 226 or a variant thereof that exhibits at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:226.

86. The immunomodulatory protein of any of embodiments 81-85, whereinthe Fc region exhibits one or more effector functions.

87. The immunomodulatory protein of any of embodiments 81-86, whereinthe Fc region exhibits one or more reduced effector function compared toa wildtype Fc region, optionally wherein the wildtype human Fc is ofhuman IgG1.

88. The immunomodulatory protein of embodiment 86 or embodiment 87,wherein the one or more effector function is selected from amongantibody dependent cellular cytotoxicity (ADCC), complement dependentcytotoxicity, programmed cell death and cellular phagocytosis.

89. The immunomodulatory protein of embodiment 87 or embodiment 88,wherein the Fc region is a variant Fc region comprising one or moreamino acid substitutions compared to the wildtype Fc region.

90. The immunomodulatory protein of embodiment 89, wherein the one ormore amino acid substitutions of the variant Fc region are selected fromN297G, E233P/L234V/L235A/G236del/S267K or L234A/L235E/G237A, wherein theresidue is numbered according to the EU index of Kabat.

91. The immunomodulatory protein of embodiment 90, wherein the variantFc region further comprises the amino acid substitution C220S, whereinthe residues are numbered according to the EU index of Kabat.

92. The immunomodulatory protein of any of embodiments 87-91, whereinthe Fc region comprises the sequence of amino acid sequence set forth inany of SEQ ID NOS: 476-478 or a sequence of amino acids that exhibits atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to any of SEQ ID NOS:476-478 and contains the amino acidsubstitutions.

93. The immunomodulatory protein of any of embodiments 87-92, whereinthe Fc region comprises K447del, wherein the residue is numberedaccording to the EU index of Kabat.

94. The immunomodulatory protein of any of embodiments 87-92 and 93,wherein the Fc region comprises the sequence of amino acid sequence setforth in any of SEQ ID NOS: 632-634 or a sequence of amino acids thatexhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to any of SEQ ID NOS:632-634 and contains theamino acid substitutions.

95. An immunomodulatory protein, comprising:

(a) a variant ICOSL polypeptide comprising one or more amino acidmodifications in an immunoglobulin superfamily (IgSF) domain of an ICOSLreference polypeptide, wherein the variant ICOSL polypeptide exhibitsaltered binding to the ectodomain(s) of ICOS or CD28 compared to thebinding of the ICOSL reference polypeptide for the same ectodomain(s);and

(b) a variant Fc region comprising amino acid substitutions selectedfrom N297G/K447del, E233P/L234V/L235A/G236del/S267K/K447del orL234A/L235E/G237A/K447del compared to wildtype human IgG1, wherein theresidues are numbered according to the EU index of Kabat.

96. The immunomodulatory protein of embodiment 95 that is a dimer.

97. The immunomodulatory protein of embodiment 95 or embodiment 96,wherein the variant Fc region further comprises the amino acidsubstitution C220S, wherein the residues are numbered according to theEU index of Kabat.

98. The immunomodulatory protein of any of embodiments 95-97, whereinthe Fc region comprises the sequence of amino acid sequence set forth inany of SEQ ID NOS: 632-634 or a sequence of amino acids that exhibits atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to any of SEQ ID NOS: 632-634 and contains the amino acidsubstitutions.

99. The immunomodulatory protein of any of embodiments 95-98, whereinthe variant ICOSL polypeptide is linked, directly or indirectly via alinker, to the variant Fc region.

100. The immunomodulatory protein of embodiment 75 and embodiment 99,wherein the linker comprises 1 to 10 amino acids.

101. The immunomodulatory protein of embodiment 100, wherein the linkeris selected from AAA, G4S (SEQ ID NO: 636), (G₄S)₂ (SEQ ID NO:229) orGSGGGGS linker (SEQ ID NO: 635).

102. An immunomodulatory protein, comprising the variant ICOSLpolypeptide of any of embodiments 1-68 linked to a second polypeptidecomprising an immunoglobulin superfamily (IgSF) domain.

103. The immunomodulatory protein of embodiment 102, wherein the IgSFdomain is affinity modified and exhibits altered binding to one or moreof its cognate binding partner(s) compared to the unmodified orwild-type IgSF domain.

104. The immunomodulatory polypeptide of embodiment 103, wherein theIgSF domain exhibits increased binding to one or more of its cognatebinding partner(s) compared to the unmodified or wild-type IgSF domain.

105. The immunomodulatory polypeptide of any one of embodiments 102-104,wherein the variant ICOSL polypeptide is a first ICOSL variantpolypeptide and the IgSF domain of the second polypeptide is an IgSFdomain from a second variant ICOSL polypeptide of any of embodiments1-68, wherein the first and second ICOSL variant are the same ordifferent.

106. The immunomodulatory protein of any one of embodiments 102-105,wherein the variant ICOSL polypeptide is capable of specifically bindingto CD28 or ICOS and the IgSF domain of the second polypeptide is capableof binding to a binding partner other than one specifically bound by theICOSL variant polypeptide.

107. The immunomodulatory polypeptide of embodiment 106, wherein theIgSF domain is from a member of the B7 family.

108. The immunomodulatory polypeptide of any of embodiments 102-104 and106, wherein the IgSF domain is a tumor-localizing moiety that binds toa ligand expressed on a tumor or is an inflammatory-localizing moietythat binds to a ligand expressed on a cell or tissue of an inflammatoryenvironment.

109. The immunomodulatory polypeptide of embodiment 108, wherein theligand is B7H6.

110. The immunomodulatory polypeptide of embodiment 108 or embodiment109, wherein the IgSF domain is from NKp30.

111. The immunomodulatory polypeptide of any of embodiments 102-110,wherein the IgSF domain is or comprises an IgV domain.

112. The immunomodulatory polypeptide of any of embodiments 102-111,wherein the variant ICOSL polypeptide is or comprise an IgV domain.

113. The immunomodulatory protein of any of embodiments 102-112, whereinthe immunomodulatory protein comprises a multimerization domain linkedto one or both of the variant ICOSL polypeptide or the secondpolypeptide comprising the IgSF domain.

114. The immunomodulatory protein of embodiment 113, wherein themultimerization domain is an Fc domain or a variant thereof with reducedeffector function.

115. The immunomodulatory protein of any of embodiments 102-114 that isdimeric.

116. The immunomodulatory protein of embodiment 115 that is homodimeric.

117. The immunomodulatory protein of embodiment 116 that isheterodimeric.

118. A conjugate comprising the variant ICOSL polypeptide of any ofembodiments 1-68 or immunomodulatory protein of any of embodiments69-117 and a heterologous moiety.

119. The conjugate of embodiment 118, wherein the variant ICOSLpolypeptide is linked, directly or indirectly via a linker, to theheterologous moiety.

120. The conjugate of any of embodiments 118 or embodiment 119, whereinthe targeting moiety is a protein, a peptide, nucleic acid, smallmolecule or nanoparticle.

121. The conjugate of any of embodiments 118-120, wherein the targetmoiety is a protein or a peptide.

122. The conjugate of embodiment 121, wherein the conjugate is a fusionprotein.

123. A fusion protein, comprising a variant ICOSL polypeptide of any ofembodiments 1-68 or immunomodulatory protein of any of embodiments69-117 and a heterologous moiety.

124. The conjugate or fusion protein of any of embodiments 118-123,wherein the moiety is a targeting moiety that specifically binds to amolecule on the surface of a cell.

125. The conjugate or fusion protein of embodiment 124, wherein thetargeting moiety specifically binds to a molecule on the surface of animmune cell.

126. The conjugate or fusion protein of embodiment 125, wherein theimmune cell is an antigen presenting cell or a lymphocyte.

127. The conjugate or fusion protein of embodiment 124, wherein thetargeting moiety is a tumor-localizing moiety that binds to a moleculeon the surface of a tumor.

128. The conjugate or fusion protein of any of embodiments 124-127,wherein the targeting moiety binds to a molecule HER1/EGFR, HER2/ERBB2,CD20, CD25 (IL-2Rα receptor), CD33, CD52, CD133, CD206, CEA, CEACAM1,CEACAM3, CEACAM5, CEACAM6, cancer antigen 125 (CA125), alpha-fetoprotein(AFP), Lewis Y, TAG72, Caprin-1, mesothelin, PDGF receptor (PDGFR),PDGF-R α, PD-1, PD-L1, CTLA-4, IL-2 receptor, vascular endothelialgrowth factor (VEGF), CD30, EpCAM, EphA2, Glypican-3, gpA33, mucins,CAIX, PSMA, folate-binding protein, gangliosides (such as GD2, GD3, GM1and GM2), VEGF receptor (VEGFR), VEGFR2, VEGF-A, integrin αVβ3, integrinα5β1, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin,AFP, BCR complex, CD3, CD18, CD44, CTLA-4, gp72, HLA-DR 10β, HLA-DRantigen, IgE, MUC-1, nuC242, PEM antigen, metalloproteinases, Ephrinreceptor, Ephrin ligands, HGF receptor, CXCR4, CXCR4, Bombesin receptor,SK-1antigen, Bcr-abl, RET, MET, TRKB, TIE2, ALK, ROS, EML4-ALK, ROS1,BRAFV600E, SRC, c-KIT, mTOR, TSC1, TSC2, BTK, KIT, BRCA, CDK 4/6, JAK1,JAK2, BRAF, FLT-3, MEK1, MEK2, SMO or B7-H6 (NCR3LG1).

129. The conjugate or fusion protein of any of embodiments 124-128,wherein the targeting moiety binds to PD-L1.

130. The conjugate or fusion protein of any of embodiments 124-129,wherein the targeting moiety is an antibody or antigen-binding fragment.

131. The conjugate or fusion protein of embodiment 130, wherein theantibody is selected from cetuximab, panitumumab, zalutumumab,nimotuzumab, trastuzumab, Ado-trastuzumab emtansine, Tositumomab(Bexxar®), Rituximab (Rituxan, Mabthera), Ibritumomab tiuxetan(Zevalin), Daclizumab (Zenapax), Gemtuzumab (Mylotarg), Alemtuzumab,CEA-scan Fab fragment, OC125 monoclonal antibody, ab75705, B72.3,Bevacizumab (Avastin®), Afatinib, Axitinib, Bosutinib, Cabozantinib,Ceritinib, Crizotinib, Dabrafenib, Dasatinib, Dinutuximab, Erlotinib,Everolimus, Ibrutinib, Imatinib, Lapatinib, Lenvatinib, Nilotinib,Olaparib, Olaratumab, Palbociclib, Pazopanib, Pertuzumab, Ramucirumab,Regorafenib, Ruxolitinib, Sorafenib, Sunitinib, Temsirolimus,Trametinib, Vandetanib, Vemurafenib, Vismodegib, Basiliximab,Ipilimumab, Nivolumab, pembrolizumab, MPDL3280A, Pidilizumab (CT-011),AMP-224, MSB001078C, or MEDI4736, BMS-935559, LY3300054, atezolizumab,avelumab or durvalumab or is an antigen-binding fragment thereof.

132. The conjugate or fusion protein of embodiment 130 or embodiment131, wherein the variant ICOSL polypeptide is linked, directly orindirectly via a linker, to the N-terminus of the heavy and/or lightchain of the antibody or antigen-binding fragment.

133. The conjugate or fusion protein of embodiment 130 or embodiment131, wherein the variant ICOSL polypeptide is linked, directly orindirectly via a linker, to the C-terminus of the heavy and/or lightchain of the antibody or antigen binding fragment.

134. The conjugate or fusion protein of any of embodiments 118-133,wherein the conjugate is divalent, tetravalent, hexavalent oroctavalent.

135. The conjugate or fusion protein of any of embodiments 118-123,wherein the heterologous moiety is or comprises a label for detection orpurification of the variant ICOSL polypeptide.

136. A monovalent fusion protein comprising:

(a) a variant ICOSL polypeptide comprising one or more amino acidmodifications in an immunoglobulin superfamily (IgSF) domain of an ICOSLreference polypeptide, wherein the variant ICOSL polypeptide exhibitsaltered binding to the ectodomain(s) of ICOS or CD28 compared to thebinding of the ICOSL reference polypeptide for the same ectodomain(s);and

(b) a label for detection or purification of the variant ICOSLpolypeptide.

137. The conjugate or fusion protein of embodiment 135 or embodiment136, wherein the label for detection or purification is selected from apoly-histidine (His) tag, a FLAG-tag, a Myc-tag, or a fluorescentprotein-tag.

138. The immunomodulatory protein of any of embodiments 95-101 or thefusion protein of embodiment 136 or embodiment 137, wherein the variantICOSL polypeptide comprises one or more amino acid modifications are ina position corresponding to position(s) selected from 10, 11, 13, 16,18, 20, 25, 26, 27, 30, 33, 37, 38, 42, 43, 47, 52, 54, 57, 61, 62, 67,71, 72, 74, 75, 77, 78, 80, 84, 89, 90, 92, 93, 94, 96, 97, 98, 99, 100,102, 103, 107, 109, 110, 111, 113, 115, 116, 117, 119, 120, 121, 122,126, 129, 130, 132, 133, 135, 137, 138, 139, 140, 142, 143, 144, 146,151, 152, 153, 154, 155, 156, 158, 161, 164, 166, 168, 172, 173, 175,190, 192, 193, 194, 198, 201, 203, 207, 208, 210, 212, 217, 218, 220,221, 224, 225, or 227 with reference to SEQ ID NO:32.

139. The immunomodulatory protein or fusion protein of embodiment 138,wherein the one or more amino acid modifications are selected from M10V,M10I, V11E, S13G, E16V, S18R, A20T, A20V, S25G, R26S, F27C, F27S, N30D,Y33del, Q37R, T38P, K42E, T43A, Y47H, N52A, N52C, N52D, N52G, N52H,N52K, N52L, N52M, N52P, N52Q, N52R, N52S, N52T, N52V, N52Y, S54A, S54F,S54P, N57A, N57D, N57E, N57F, N57H, N57K, N57L, N57M, N57P, N57Q, N57S,N57T, N57V, N57W, N57Y, R61C, R61S, Y62F, L67P, A71T, G72R, L74Q, R75Q,D77G, F78L, L80P, N84Q, D89G, E90A, K92R, F93L, H94D, H94E, L96F, L96I,V97A, L98F, S99G, Q100A, Q100D, Q100E, Q100G, Q100K, Q100L, Q100M,Q100N, Q100P, Q100R, Q100S, Q100T, Q100V, L102R, G103E, V107A, V107I,S109G, S109N, V110A, V110D, V110N, E111del, T113E, H115Q, H115R, V116A,A117T, N119Q, F120I, F120S, S121G, V122A, V122M, S126R, S126T, H129P,S130G, S132F, Q133H, E135K, T137A, F138L, T139S, C140del, C140D, S142F,I143T, I143V, N144D, Y146C, V151A, Y152C, Y152H, W153R, I154F, N155H,N155Q, K156M, D158G, L161M, L161P, Q164L, L166Q, N168Q, F172S, L173S,M175T, T190A, T190S, S192G, V193A, V193M, N194D, C198R, N201S, L203F,L203P, N207Q, L208P, V210A, S212G, D217G, D217V, I218N, I218T, E220G,R221G, R221I, R221K, I224V, T225A, T225S, N227K, or a conservative aminoacid substitution thereof.

140. The immunomodulatory protein or fusion protein of embodiment 138 orembodiment 139, wherein the ICOSL reference polypeptide comprises (i)the sequence of amino acids set forth in SEQ ID NO:32, (ii) a sequenceof amino acids that has at least 95% sequence identity to SEQ ID NO:32;or (iii) a portion of (i) or (ii) comprising an IgV domain or IgC domainor specific binding fragments thereof or both.

141. The immunomodulatory protein or fusion protein of any ofembodiments 138-140, wherein the ICOSL reference polypeptide comprisesthe sequence of amino acids set forth in any of SEQ ID NOS: 196, 545,600-605 and 623-628.

142. The immunomodulatory protein or fusion protein of any ofembodiments 138-141, wherein the ICOSL reference polypeptide consists ofthe sequence of amino acids set forth in any of SEQ ID NOS: 32, 196,545, 600-605 and 623-628.

143. A nucleic acid molecule(s), encoding a variant ICOSL polypeptide ofany of embodiments 1-68, an immunomodulatory protein of any ofembodiments 69-117 and 118-142 or a fusion protein of any of embodiments123-142.

144. The nucleic acid molecule(s) of embodiment 143 that is syntheticnucleic acid.

145. The nucleic acid molecule(s) of embodiment 143 or embodiment 144that is cDNA.

146. A vector, comprising the nucleic acid molecule(s) of any ofembodiments 143-145.

147. The vector of embodiment 146 that is an expression vector.

148. The vector of embodiment 146 or embodiment 147, wherein the vectoris a mammalian expression vector or a viral vector.

149. A cell, comprising the vector of any of embodiments 146-148.

150. The cell of embodiment 149 that is a mammalian cell.

151. The cell of embodiment 149 or embodiment 150 that is a ChineseHamster Ovary (CHO) cell or a derivative thereof.

152. A method of producing an immunomodulatory protein comprising avariant ICOSL polypeptide, comprising introducing the nucleic acidmolecule of any of embodiments 143-145 or vector of any of embodiments146-148 into a host cell under conditions to express the protein in thecell.

153. The method of embodiment 152, wherein the host cell is a mammaliancell.

154. The method of embodiment 153, wherein the mammalian cell is aChinese Hamster Ovary cell or a derivative thereof.

155. The method of any of embodiments 152-154, further comprisingisolating or purifying the protein from the cell.

156. A protein produced by the method of any of embodiments 152-155.

157. A composition comprising a protein comprising a variant ICOSLpolypeptide of any of embodiments 1-68 or an immunomodulatory protein ofany of embodiments 69-117, wherein at least 95%, 96%, 97%, 98%, 99% ofthe individual sequences of the protein or the immunomodulatory proteinin the composition have an identical sequence length, optionally whereinthe composition is a pharmaceutical composition comprising apharmaceutically acceptable carrier.

158. The composition of embodiment 157, wherein the protein orimmunomodulatory protein is purified from Chinese Hamster Ovary Cells ora derivative thereof.

159. A polynucleotide comprising a nucleic acid encoding a variant ICOSLpolypeptide comprising a transmembrane domain of any of embodiments64-68 and one or more nucleic acid encoding one or more chain of arecombinant antigen receptor.

160. The polynucleotide of embodiment 159, wherein the recombinantantigen receptor is a chimeric antigen receptor (CAR) or an engineered Tcell receptor (TCR).

161. The polynucleotide of embodiment 159 or embodiment 160, whereineach of the nucleic acid encoding the variant ICOSL polypeptide and theone or more nucleic acid encoding one or more chain of the recombinantreceptor is separated by a nucleic acid encoding a self-cleaving peptideor a peptide that causes ribosome skipping.

162. The polynucleotide of embodiment 161, wherein the polynucleotidecomprises the nucleic acid encoding the variant ICOSL polypeptide, anucleic acid encoding a self-cleaving peptide or a peptide that causesribosome skipping and a nucleic acid encoding a CAR.

163. The polynucleotide of embodiment 161, wherein the polynucleotidecomprises the nucleic acid encoding the variant ICOSL polypeptide, anucleic acid encoding a first self-cleaving peptide or a peptide thatcauses ribosome skipping, a nucleic acid encoding one of an engineeredTCRalpha chain or an engineered TCRbeta chain, a nucleic acid encoding asecond self-cleaving peptide or a peptide that causes ribosome skipping,and a nucleic acid encoding the other of the engineered TCRalpha chainor the engineered TCRbeta chain.

164. The polynucleotide of embodiment 163, wherein the encoded first andsecond self-cleaving peptide is the same.

165. The polynucleotide of any of embodiments 160-163, wherein theself-cleaving peptide or the peptide that causes ribosome skipping is aT2A, a P2A, a E2A or a F2A.

166. A vector comprising the polynucleotide of any of embodiments159-165.

167. The vector of embodiment 166, wherein the vector is a viral vector.

168. The vector of embodiment 167, wherein the viral vector is aretroviral vector or a lentiviral vector.

169. An engineered cell comprising the polynucleotide of any ofembodiments 159-165 or the vector of any of embodiments 166-168.

170. An engineered cell comprising the variant ICOSL polypeptide of anyof embodiments 1-68, the immunomodulatory protein of any of embodiments69-117, or the fusion protein of any of embodiments 123-142.

171. An engineered cell comprising the nucleic acid molecule of any ofembodiments 143-145 or the vector of any of embodiments 146-148.

172. The engineered cell of any of embodiments 169-171, wherein thenucleic acid encoding the variant ICOSL polypeptide, immunomodulatoryprotein or fusion protein encodes a signal peptide.

173. The engineered cell of any of embodiments 169-172, wherein thevariant ICOSL polypeptide, immunomodulatory protein or fusion proteindoes not comprise a transmembrane domain and/or is not expressed on thesurface of the cell.

174. The engineered cell of any of embodiments 169-173, wherein thevariant ICOSL polypeptide, immunomodulatory protein or fusion protein issecreted from the engineered cell.

175. The engineered cell of any of embodiments 169-171, wherein theengineered cell comprises a variant ICOSL polypeptide comprising atransmembrane domain of any of embodiments 64-68.

176. The engineered cell of any of embodiments 169-171 and 175, whereinthe variant ICOSL polypeptide is expressed on the surface of the cell.

177. The engineered cell of any of embodiments 169-176, wherein the cellis an immune cell.

178. The engineered cell of embodiment 177, wherein the immune cell isan antigen presenting cell (APC) or a lymphocyte.

179. The engineered cell of any of embodiments 169-178 that is a primarycell.

180. The engineered cell of any of embodiments 169-179, wherein the cellis a mammalian cell.

181. The engineered cell of any of embodiments 169-180, wherein the cellis a human cell.

182. The engineered cell of any of embodiments 169-181, wherein thelymphocyte is a T cell.

183. The engineered cell of embodiment 178, wherein the engineered cellis an APC and the APC is an artificial APC.

184. The engineered cell of any of embodiments 169-183, furthercomprising a chimeric antigen receptor (CAR) or an engineered T-cellreceptor.

185. A pharmaceutical composition, comprising the variant ICOSLpolypeptide of any of embodiments 1-68, the immunomodulatory protein ofany of embodiments 69-117, a conjugate or fusion protein of any ofembodiments 118-142 or an engineered cell of any of embodiments 169-184or an infectious agent of an of embodiments 216-227.

186. The pharmaceutical composition of embodiment 185, comprising apharmaceutically acceptable excipient.

187. The pharmaceutical composition of embodiment 185 or 186, whereinthe pharmaceutical composition is sterile.

188. An article of manufacture comprising the pharmaceutical compositionof any of embodiments 185-187 in a vial.

189. The article of manufacture of embodiment 188, wherein the vial issealed.

190. A kit comprising the composition of any of embodiments 157-158 and185-187, and instructions for use.

191. A kit comprising the article of manufacture according to embodiment189 and 190, and instructions for use.

192. A method of modulating an immune response in a subject, comprisingadministering the pharmaceutical composition of any of embodiments157-158 and 185-187 to the subject.

193. A method of modulating an immune response in a subject, comprisingadministering the engineered cells of any of embodiments 169-184.

194. The method of embodiment 193, wherein the engineered cells areautologous to the subject.

195. The method of embodiment 193, wherein the engineered cells areallogenic to the subject.

196. The method of any of embodiments 193-195, wherein modulating theimmune response treats a disease or condition in the subject.

197. The method of any of embodiments 193-196, wherein the immuneresponse is increased.

198. The method of any of embodiments 192, 196 and 197, wherein animmunomodulatory protein or conjugate comprising a variant ICOSLpolypeptide linked to a tumor-localizing moiety is administered to thesubject.

199. The method of embodiment 198, wherein the tumor-localizing moietyis or comprises a binding molecule that recognizes a tumor antigen.

200. The method of embodiment 199, wherein the binding moleculecomprises an antibody or an antigen-binding fragment thereof orcomprises a wild-type IgSF domain or variant thereof.

201. The method of any of embodiments 192 and 196-200, wherein theimmunomodulatory protein of any of embodiments 102-117 or the conjugateor fusion protein of any of embodiments 118-142 is administered to thesubject.

202. The method of any of embodiments 193-197, wherein a variant ICOSLpolypeptide that is a transmembrane immunomodulatory protein isadministered to the subject.

203. The method of any of embodiments 193-197 and 202, wherein theengineered cell comprising a variant ICOSL polypeptide that is atransmembrane immunomodulatory protein of any of embodiments 64-68 isadministered to the subject.

204. The method of embodiment 192-203, wherein the disease or conditionis a tumor or cancer.

205. The method of any one of embodiments 192-204, wherein the diseaseor condition is selected from melanoma, lung cancer, bladder cancer, ahematological malignancy, liver cancer, brain cancer, renal cancer,breast cancer, pancreatic cancer, colorectal cancer, spleen cancer,prostate cancer, testicular cancer, ovarian cancer, uterine cancer,gastric carcinoma, a musculoskeletal cancer, a head and neck cancer, agastrointestinal cancer, a germ cell cancer, or an endocrine andneuroendocrine cancer.

206. The method of any of embodiments 192-196, wherein the immuneresponse is decreased.

207. The method of any of embodiments 192-196 and 206, wherein a variantICOSL polypeptide or immunomodulatory protein that is soluble isadministered to the subject.

208. The method of embodiment 207, wherein the soluble immunomodulatoryprotein is an immunomodulatory Fc fusion protein.

209. The method of any of embodiments 192-196 and 206-208, wherein avariant ICOSL polypeptide of any of embodiments 1-63 and 68, theimmunomodulatory protein of any of embodiments 70-101 or the fusionprotein of embodiment 136 and 137 is administered to the subject.

210. The method of any of embodiments 192-196 and 206-208, wherein anengineered cell comprising a secretable variant ICOSL polypeptide isadministered to the subject.

211. The method of any of embodiments 192-196, 206-208 and 210, whereinan engineered cell of any of embodiments 169-174 and 177-184 isadministered to the subject.

212. The method of any of embodiments 192-196, 206-208 and 210, whereinan infectious agent encoding a variant ICOSL polypeptide that is asecretable immunomodulatory protein is administered to the subject,optionally under conditions in which the infectious agent infects atumor cell or immune cell and the secretable immunomodulatory protein issecreted from the infected cell.

213. The method of any of embodiments 192-196 and 206-212, wherein thedisease or condition is an inflammatory or autoimmune disease orcondition.

214. The method of any of embodiments 192-196 and 206-213, wherein thedisease or condition is an Antineutrophil cytoplasmic antibodies(ANCA)-associated vasculitis, a vasculitis, an autoimmune skin disease,transplantation, a Rheumatic disease, an inflammatory gastrointestinaldisease, an inflammatory eye disease, an inflammatory neurologicaldisease, an inflammatory pulmonary disease, an inflammatory endocrinedisease, or an autoimmune hematological disease.

215. The method of embodiment 213 or embodiment 214, wherein the diseaseor condition is selected from inflammatory bowel disease, transplant,Crohn's disease, ulcerative colitis, multiple sclerosis, asthma,rheumatoid arthritis, or psoriasis.

216. An infectious agent, comprising a nucleic acid molecule encoding avariant ICOSL polypeptide of any of embodiments 1-68 or animmunomodulatory protein of any of embodiments the immunomodulatoryprotein of any of embodiments 69-117, or the fusion protein of any ofembodiments 123-142.

217. The infectious agent of embodiment 216, wherein the encoded variantICOSL polypeptide, immunomodulatory protein or fusion protein does notcomprise a transmembrane domain and/or is not expressed on the surfaceof a cell in which it is expressed.

218. The infectious agent of embodiment 216 or embodiment 217, whereinthe encoded variant ICOSL polypeptide, immunomodulatory protein orfusion protein is secreted from the infectious agent when it isexpressed.

219. The infectious agent of embodiment 218, wherein the encoded variantICOSL polypeptide comprises a transmembrane domain.

220. The infectious agent of embodiment 216, embodiment 217 orembodiment 219, wherein the encoded variant ICOSL polypeptide isexpressed on the surface of a cell in which it is expressed.

221. The infectious agent of any of embodiments 216-220, wherein theinfectious agent is a bacteria or a virus.

222. The infectious agent of embodiment 221, wherein the virus is anoncolytic virus.

223. The infectious agent of embodiment 222, wherein the oncolytic virusis an adenoviruses, adeno-associated viruses, herpes viruses, HerpesSimplex Virus, Vesticular Stomatic virus, Reovirus, Newcastle Diseasevirus, parvovirus, measles virus, vesticular stomatitis virus (VSV),Coxsackie virus or a Vaccinia virus.

224. The infectious agent of embodiment 222, wherein the virusspecifically targets dendritic cells (DCs) and/or is dendriticcell-tropic.

225. The infectious agent of embodiment 224, wherein the virus is alentiviral vector that is pseudotyped with a modified Sindbis virusenvelope product.

226. The infectious agent of any of embodiments 216-225, furthercomprising a nucleic acid molecule encoding a further gene product thatresults in death of a target cell or that can augment or boost an immuneresponse.

227. The infectious agent of embodiment 226, wherein the further geneproduct is selected from an anticancer agent, anti-metastatic agent, anantiangiogenic agent, an immunomodulatory molecule, an immune checkpointinhibitor, an antibody, a cytokine, a growth factor, an antigen, acytotoxic gene product, a pro-apoptotic gene product, an anti-apoptoticgene product, a cell matrix degradative gene, genes for tissueregeneration or a reprogramming human somatic cells to pluripotency.

X. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 Example 1 Generation of Mutant DNA Constructs of IgSF Domains

Example 1 describes the generation of mutant DNA constructs of humanICOSL IgSF domains for translation and expression on the surface ofyeast as yeast display libraries.

A. Degenerate Libraries

Mutant DNA constructs encoding a variant of the ECD domain of ICOSL weregenerated. Constructs were generated based on a wildtype human ICOSLsequence set forth in SEQ ID NO:32 containing the ECD domain as follows:

DTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIENVLLQQNLTVGSQTGNDIGERDKITENPVSTGEKNAAT

For libraries that target specific residues for complete or partialrandomization with degenerate codons, the DNA encoding SEQ ID NO:32 wasordered from Integrated DNA Technologies (Coralville, Iowa) as a set ofoverlapping oligonucleotides of up to 80 base pairs (bp) in length. Togenerate a library of diverse variants of the ECD, the oligonucleotidescontained desired degenerate codons, such as specific mixed base sets tocode for various amino acid substitutions, at desired amino acidpositions. Degenerate codons were generated using an algorithm at theURL: rosettadesign.med.unc.edu/SwiftLib/.

In general, positions to mutate and degenerate codons were chosen fromhomology models (ICOSL) of the target-ligand pairs of interest toidentify ligand contact residues, such as target side chain residuesthat interact with the ligand, as well as residues that are at theprotein interaction interface. This analysis was performed using astructure viewer available at the URL: spdbv.vital-it.ch).

The next step in library design was the alignment of human, mouse, ratand monkey ICOSL sequences to identify conserved residues. Based on thisanalysis, conserved target residues were mutated with degenerate codonsthat only specified conservative amino acid changes plus the wild-typeresidue. Residues that were not conserved, were mutated moreaggressively, but also included the wild-type residue. Degenerate codonsthat also encoded the wild-type residue were deployed to avoid excessivemutagenesis of target protein. For the same reason, only up to 20positions were targeted for mutagenesis at a time. These residues were acombination of contact residues and non-contact interface residues.

The oligonucleotides were dissolved in sterile water, mixed in equimolarratios, heated to 95° C. for five minutes and slowly cooled to roomtemperature for annealing. ECD-specific oligonucleotide primers thatanneal to the start and end of the ECDs, respectively, were then used togenerate PCR product. ECD-specific oligonucleotides which overlap by40-50 bp with a modified version of pBYDS03 cloning vector (LifeTechnologies USA), beyond and including the BamH1 and Kpn1 cloningsites, were then used to amplify 100 ng of PCR product from the priorstep to generate a total of 5 μg of DNA. Both PCR's were by polymerasechain reaction (PCR) using OneTaq 2×PCR master mix (New England Biolabs,USA). The second PCR products were purified using a PCR purification kit(Qiagen, Germany) and resuspended in sterile deionized water.

To prepare for library insertion, a modified yeast display version ofvector pBYDS03 was digested with BamHI and KpnI restriction enzymes (NewEngland Biolabs, USA) and the large vector fragment was gel-purified anddissolved in sterile, deionized water. Electroporation-ready DNA for thenext step was generated by mixing 12 μg of library DNA for everyelectroporation with 4 μg of linearized vector in a total volume of 50μl deionized and sterile water. An alternative way to generate targetedlibraries, was to carry out site-directed mutagenesis (Multisite kit,Agilent, USA) of the target ECD with oligonucleotides containingdegenerate codons. This approach was used to generate sublibraries thatonly target specific stretches of target protein for mutagenesis. Inthese cases, sublibraries were mixed before proceeding to the selectionsteps. In general, library sizes were in the range of 10E7 to 10E8clones, except that sublibraries were only in the range of 10E4 to 10E5.Large libraries and sublibraries are generated for ICOSL.

B. Random Libraries

Random libraries were also constructed to identify variants of the ECDof ICOSL set forth in SEQ ID NO:32 containing the ECD domain. DNAencoding the wild-type ECD was cloned between the BamHI and KpnIrestriction sites of modified yeast display vector pBYDS03. The DNA wasthen mutagenized with the Genemorph II Kit (Agilent, USA) to generate anaverage of three to five amino acid changes per library variant.Mutagenized DNA was then amplified by the two-step PCR and furtherprocessed as described above for targeted libraries.

Example 2 Introduction of DNA Libraries into Yeast

Example 2 describes the introduction of ICOSL DNA libraries into yeast.

To introduce degenerate and random library DNA into yeast,electroporation-competent cells of yeast strain BJ5464 (ATCC.org; ATCCnumber 208288) were prepared and electroporated on a Gene Pulser II(Biorad, USA) with the electroporation-ready DNA from the step aboveessentially as described (Colby, D. W. et al. 2004 Methods Enzymology388, 348-358). The only exception is that transformed cells were grownin non-inducing minimal selective SCD-Leu medium to accommodate the LEU2selectable marker carried by modified plasmid pBYDS03. One liter ofSCD-Leu media consists of 14.7 grams sodium citrate, 4.29 grams citricacid monohydrate, 20 grams dextrose, 6.7 grams yeast nitrogen base, and1.6 grams yeast synthetic drop-out media supplement without leucine. TheMedium was filter sterilized before use using a 0.22 μm vacuum filterdevice.

Library size was determined by plating serial dilutions of freshlyrecovered cells on SCD-Leu agar plates and then extrapolating librarysize from the number of single colonies from plating that generated atleast 50 colonies per plate. In general, library sizes ranged from 10E8to 10E9 transformants based on this dilution assay. The remainder of theelectroporated culture was grown to saturation in SCD-Leu and cells fromthis culture were subcultured (e.g. 1/100) into fresh SCD-Leu once moreto minimize the fraction of untransformed cells. To maintain librarydiversity, this subculturing step was carried out using an inoculum thatcontained at least 10× more cells than the calculated library size.Cells from the second saturated culture were resuspended in fresh mediumcontaining sterile 25% (weight/volume) glycerol to a density of 10E10/mLand frozen and stored at −80C (frozen library stock).

Library size was determined by plating dilutions of freshly recoveredcells on SCD-Leu agar plates and then extrapolating library size fromthe number of single colonies from a plating that generate at least 50colonies per plate.

To segregate plasmid from cells that contain two or more differentlibrary clones, a number of cells corresponding to 10 times the librarysize, were taken from the overnight SCD-Leu culture and subcultured1/100 into fresh SCD-Leu medium and grown overnight. Cells from thisovernight culture were resuspended in sterile 25% (weight/volume)glycerol to a density of 10E10/mL and frozen and stored at −80C (frozenlibrary stock).

Example 3 Yeast Selection

Example 3 describes the selection of yeast expressing affinity modifiedvariants of ICOSL.

A number of cells equal to at least 10 times the library size werethawed from individual library stocks, suspended to 0.1×10E6 cells/mL innon-inducing SCD-Leu medium, and grown overnight. The next day, a numberof cells equal to 10 times the library size were centrifuged at 2000 RPMfor two minutes and resuspended to 0.5×10E6 cells/mL in inducingSCDG-Leu media. One liter of the SCDG-Leu induction media consists of5.4 grams Na₂HPO₄, 8.56 grams of NaH₂PO₄.H₂0, 20 grams galactose, 2.0grams dextrose, 6.7 grams Difco yeast nitrogen base, and 1.6 grams ofyeast synthetic drop out media supplement without leucine dissolved inwater and sterilized through a 0.22 μm membrane filter device. Theculture was grown for two days at 20° C. to induce expression of libraryproteins on the yeast cell surface.

Cells were processed with magnetic beads to reduce non-binders andenrich for all ICOSL variants with the ability to bind their exogenousrecombinant counter-structure proteins. This was then followed by two tothree rounds of flow cytometry sorting using exogenous counter-structureprotein staining to enrich the fraction of yeast cells that displaysimproved binders. Magnetic bead enrichment and selections by flowcytometry are essentially as described in Miller, K. D. CurrentProtocols in Cytometry 4.7.1-4.7.30, July 2008.

With ICOSL libraries, target ligand proteins were sourced from R&DSystems (USA) as follows: human rCD28.Fc (i.e., recombinant CD28-Fcfusion protein), rCTLA4.Fc and rICOS.Fc. Magnetic streptavidin beadswere obtained from New England Biolabs, USA. For biotinylation ofcounter-structure protein, biotinylation kit cat #21955, LifeTechnologies, USA, was used. For two-color, flow cytometric sorting, aBecton Dickinson FACS Aria II sorter was used. ICOSL display levels weremonitored with an anti-hemagglutinin antibody labeled with Alexafluor488 (Life Technologies, USA). Ligand binding Fc fusion proteinsrCD28.Fc, rCTLA4.Fc, or rICOS.Fc were detected with PE conjugated humanIg specific goat Fab (Jackson ImmunoResearch, USA). Doublet yeast weregated out using forward scatter (FSC)/side scatter (SSC) parameters, andsort gates were based upon higher ligand binding detected in FL4 thatpossessed more limited tag expression binding in FL1.

Yeast outputs from the flow cytometric sorts were assayed for higherspecific binding affinity. Sort output yeast were expanded andre-induced to express the particular IgSF affinity modified domainvariants they encode. This population then can be compared to theparental, wild-type yeast strain, or any other selected outputs, such asthe bead output yeast population, by flow cytometry.

For ICOSL, the second sort outputs (F2) were compared to parental ICOSLyeast for binding of each rICOS.Fc, rCD28.Fc, and rCTLA4.Fc by doublestaining each population with anti-HA (hemagglutinin) tag expression andthe anti-human Fc secondary to detect ligand binding.

In the case of ICOSL yeast variants selected for binding to ICOS, the F2sort outputs gave Mean Fluorescence Intensity (MFI) values of 997, whenstained with 5.6 nM rICOS.Fc, whereas the parental ICOSL strain MFI wasmeasured at 397 when stained with the same concentration of rICOS.Fc.This represents a roughly three-fold improvement of the average bindingin this F2 selected pool of clones, and it is predicted that individualclones from that pool will have much better improved MFI/affinity whenindividually tested.

In the case of ICOSL yeast variants selected for binding to CD28, the F2sort outputs gave MFI values of 640 when stained with 100 nM rCD28.Fc,whereas the parental ICOSL strain MFI was measured at 29 when stainedwith the same concentration of rCD28.Fc (22-fold improvement). In thecase of ICOSL yeast variants selected for binding to CTLA4, the F2 sortoutputs gave MFI values of 949 when stained with 100 nM rCTLA4.Fc,whereas the parental ICOSL strain MFI was measured at 29 when stainedwith the same concentration of rCTLA4.Fc (32-fold improvement).

Importantly, the MFIs of all F2 outputs described above when measuredwith the anti-HA tag antibody on FL1 did not increase and sometimes wentdown compared to wild-type strains, indicating that increased bindingwas not a function of increased expression of the selected variants onthe surface of yeast, and validated gating strategies of only selectingmid to low expressors with high ligand binding.

Selected variant ICOSL ECD domains were further formatted as fusionproteins and tested for binding and functional activity as describedbelow.

Example 4 Reformatting Selection Outputs as Fc-Fusions and in VariousImmunomodulatory Protein Types

Example 4 describes reformatting of selection outputs identified inExample 3 as immunomodulatory proteins containing an affinity modified(variant) extracellular domain (ECD) of ICOSL fused to an Fc molecule(variant ECD-Fc fusion molecules).

Output cells from final flow cytometric ICOSL sorts were grown toterminal density in SCD-Leu medium. Plasmid DNA from each output wasisolated using a yeast plasmid DNA isolation kit (Zymo Research, USA).For Fc fusions, PCR primers with added restriction sites suitable forcloning into the Fc fusion vector of choice were used to batch-amplifyfrom the plasmid DNA preps the coding DNA's for the mutant target ECD's.After restriction digestion, the PCR products were ligated into anappropriate Fc fusion vector followed by chemical transformation intostrain E. coli XL1 Blue (Agilent, USA) or NEB5alpha (New EnglandBiolabs, USA) as directed by supplier. Exemplary of an Fc fusion vectoris pFUSE-hIgG1-Fc2 (InvivoGen, USA).

Dilutions of transformation reactions were plated on LB-agar containing100 μg/mL carbenicillin (Teknova, USA) to generate single colonies. Upto 96 colonies from each transformation were then grown in 96 wellplates to saturation overnight at 37° C. in LB-broth (Teknova cat #L8112) and a small aliquot from each well was submitted for DNAsequencing of the ECD insert in order to identify the mutation(s) in allclones. Sample preparation for DNA sequencing was carried out usingprotocols provided by the service provider (Genewiz; South Plainfield,N.J.). After removal of sample for DNA sequencing, glycerol was thenadded to the remaining cultures for a final glycerol content of 25% andplates were stored at −20° C. for future use as master plates (seebelow). Alternatively, samples for DNA sequencing were generated byreplica plating from grown liquid cultures onto solid agar plates usinga disposable 96 well replicator (VWR, USA). These plates were incubatedovernight to generate growth patches and the plates were submitted toGenewiz for DNA sequencing following their specifications. In someinstances, resequencing was performed to verify mutations.

After analysis of the Genewiz-generated DNA sequencing data, clones ofinterest were recovered from master plates and individually grown tosaturation in 5 mL liquid LB-broth containing 100 μg/mL carbenicillin(Teknova, USA) and 2 mL of each culture were then used for preparationof approximately 10 μg of miniprep plasmid DNA of each clone using astandard kit such as the Pureyield kit (Promega, USA). Identification ofclones of interest generally involved the following steps. First, DNAsequence data files were downloaded from the Genewiz website. Allsequences were then manually curated so that they start at the beginningof the ECD coding region. The curated sequences were thenbatch-translated using a suitable program available at the URL:www.ebi.ac.uk/Tools/st/emboss_transeq/. The translated sequences werethen aligned using a suitable program available at the URL:multalin.toulouse.inra.fr/multalin/multalin.html. Alternatively, Genewizsequences were processed to generate alignments using Ugene software(http://ugene.net).

Clones of interest were then identified using the following criteria:1.) identical clone occurs at least two times in the alignment and 2.) amutation occurs at least two times in the alignment and preferably indistinct clones. Clones that meet at least one of these criteria wereclones that have been enriched by the sorting process most likely due toimproved binding.

To generate recombinant immunomodulatory proteins that are Fc fusionproteins containing an ECD of ICOSL with at least one affinity-modifieddomain (e.g. variant ICOSL ECD-Fc), the encoding nucleic acid moleculewas generated to encode a protein designed as follows: signal peptidefollowed by variant (mutant) ICOSL ECD followed by a linker of threealanines (AAA) followed by a human IgG1 Fc containing the mutation N82Gwith reference to wild-type human IgG1 Fc set forth in SEQ ID NO: 226(corresponding to N297G by EU numbering). This exemplary Fc alsocontained stabilizing cysteine mutations R77C and V87C and replacementof the cysteine residue to a serine residue at position 220 (C220S) byEU numbering (corresponding to position 5 (C5S) with reference withreference to wild-type human IgG1 Fc set forth in SEQ ID NO:226(corresponding to R292C, V302C and C220S, respectively, by EUnumbering). In some cases, the NotI cloning site which contributes tothe AAA linker sequence was deleted to generate a direct fusion of theICOSL ECD and the beginning of the Fc. Since the construct does notinclude any antibody light chains that can form a covalent bond with acysteine, the human IgG1 Fc also contains replacement of the cysteineresidues to a serine residue at position 5 (C5S) compared to thewild-type or unmodified Fc set forth in SEQ ID NO: 226.

Example 5 Expression and Purification of Fc-Fusions

Example 5 describes the high throughput expression and purification ofFc-fusion proteins containing variant ECD ICOSL as described in theabove Examples.

Recombinant variant Fc fusion proteins were produced fromsuspension-adapted human embryonic kidney (HEK) 293 cells using theExpi293 expression system (Invitrogen, USA). 4 μg of each plasmid DNAfrom the previous step was added to 200 μLOpti MEM (Invitrogen, USA) atthe same time as 10.8 μL ExpiFectamine was separately added to another200 μL Opti-MEM. After 5 minutes, the 200 μL of plasmid DNA was mixedwith the 200 μL of ExpiFectamine and was further incubated for anadditional 20 minutes before adding this mixture to cells. Ten millionExpi293 cells were dispensed into separate wells of a sterile 10 ml,conical bottom, deep 24 well growth plate (Thomson Instrument Company,USA) in a volume 3.4 ml Expi293 media (Invitrogen, USA). Plates wereshaken for 5 days at 120 RPM in a mammalian cell culture incubator setto 95% humidity and 8% CO₂. Following a 5 day incubation, cells werepelleted and culture supernatants were retained.

Proteins were purified from supernatants using a high throughput 96 wellProtein A purification kit using the manufacturer's protocol (Catalognumber 45202, Life Technologies, USA). Resulting elution fractions werebuffer exchanged into PBS using Zeba 96 well spin desalting plate(Catalog number 89807, Life Technologies, USA) using the manufacturer'sprotocol. Purified protein was quantitated using 280 nm absorbancemeasured by Nanodrop instrument (Thermo Fisher Scientific, USA), andprotein purity was assessed by loading 5 μg of protein on NUPAGEpre-cast, polyacrylamide gels (Life Technologies, USA) under denaturingand reducing conditions and subsequent gel electrophoresis. Proteinswere visualized in gel using standard Coomassie staining.

Example 6 Assessment of Binding and Activity of Affinity-Matured IgSFDomain-Containing Molecules

A. Binding to Cell-Expressed Counter Structures

This Example describes Fc-fusion binding studies of purified proteinsfrom the above Examples to assess specificity and affinity of ICOSLdomain variant immunomodulatory proteins for cognate binding partners.

To produce cells expressing cognate binding partners, full-lengthmammalian surface expression constructs for each of human CD28 and ICOSwere designed in pcDNA3.1 expression vector (Life Technologies) andsourced from Genscript, USA. Binding studies were carried out ontransfected HEK293 cells generated to express the full-length mammaliansurface ligands using the transient transfection system (LifeTechnologies, USA) described above. As a control, binding to mock(non-transfected) cells also was assessed. The number of cells neededfor the experiment was determined, and the appropriate 30 mL scale oftransfection was performed using the manufacturer's suggested protocol.For each CD28, ICOS or mock 30 mL transfection, 75 million Expi293Fcells were incubated with 30 μg expression construct DNA and 1.5 mldiluted ExpiFectamine 293 reagent for 48 hours, at which point cellswere harvested for staining.

For staining by flow cytometry, 200,000 cells of appropriate transienttransfection or negative control (mock) were plated in 96 well roundbottom plates. Cells were spun down and resuspended in staining buffer(PBS (phosphate buffered saline), 1% BSA (bovine serum albumin), and0.1% sodium azide) for 20 minutes to block non-specific binding.Afterwards, cells were centrifuged again and resuspended in stainingbuffer containing 100 nM to InM variant immunomodulatory protein,depending on the experiment of each candidate CD80 variant Fc, ICOSLvariant Fc, or stacked IgSF variant Fc fusion protein in 50 μL. Primarystaining was performed on ice for 45 minutes, before washing cells instaining buffer twice. PE-conjugated anti-human Fc (JacksonImmunoResearch, USA) was diluted 1:150 in 50 μL staining buffer andadded to cells and incubated another 30 minutes on ice. Secondaryantibody was washed out twice, cells were fixed in 4% formaldehyde/PBS,and samples were analyzed on FACScan flow cytometer (Becton Dickinson,USA) or a Hypercyt flow cytometer (Intellicyte, USA).

Mean Fluorescence Intensity (MFI) was calculated for each transfectantand negative parental line with Cell Quest Pro software (BectonDickinson, USA) or a Hypercyt flow cytometer (Intellicyte, USA).

B. Bioactivity Characterization

This Example further describes Fc-fusion variant protein bioactivitycharacterization in human primary T cell in vitro assays.

1. Mixed Lymphocyte Reaction (MLR)

Soluble rICOSL.Fc bioactivity was tested in a human Mixed LymphocyteReaction (MLR). Human primary dendritic cells (DC) were generated byculturing monocytes isolated from PBMC (BenTech Bio, USA) in vitro for 7days with 500U/mL rIL-4 (R&D Systems, USA) and 250 U/mL rGM-CSF (R&DSystems, USA) in Ex-Vivo 15 media (Lonza, Switzerland). 10,000 maturedDC and 100,000 purified allogeneic CD4+ T cells (BenTech Bio, USA) wereco-cultured with ICOSL variant Fc fusion proteins and controls in 96well round bottom plates in 200 μl final volume of Ex-Vivo 15 media. Onday 5, IFN-gamma secretion in culture supernatants was analyzed usingthe Human IFN-gamma Duoset ELISA kit (R&D Systems, USA). Optical densitywas measured by VMax ELISA Microplate Reader (Molecular Devices, USA)and quantitated against titrated rIFN-gamma standard included in theIFN-gamma Duo-set kit (R&D Systems, USA). A second MLR protocolconsisted of human primary dendritic cells (DC) generated by culturingmonocytes isolated from PBMC (BenTech Bio, USA) in vitro for 7 days with50 ng/mL rIL-4 (R&D Systems, USA) and 80 ng/mL rGM-CSF (R&D Systems,USA) in Ex-Vivo 15 media (Lonza, Switzerland). On days 3 and 5, half ofthe media was removed and replaced with fresh media containing 50 ng/mLrIL-4 and 80 ng/mL rGM-CSF. To fully induce DC maturation,lipopolysaccharide (LPS) (InvivoGen Corp., USA) was added at 100 ng/mLto the DC cultures on day 6 and cells were incubated for an additional24 hours. Approximately, 10,000 matured DC and 100,000 purifiedallogeneic CD3+ T cells (BenTech Bio, USA) were co-cultured with ICOSLvariant Fc fusion proteins and controls in 96 well round bottom platesin 200 μl final volume of Ex-Vivo 15 media. On day 4-5, IFN-gammasecretion in culture supernatants was analyzed using the Human IFN-gammaDuoset ELISA kit (R&D Systems, USA). Optical density was measured on aBioTek Cytation Multimode Microplate Reader (BioTek Corp., USA) andquantitated against titrated rIFN-gamma standard included in theIFN-gamma Duo-set kit (R&D Systems, USA).

2. Anti-CD3 Coimmobilization Assay

Costimulatory bioactivity of ICOSL fusion variants was determined inanti-CD3 coimmobilization assays. InM or 10 nM mouse anti-human CD3(OKT3, Biolegends, USA) was diluted in PBS with InM to 80 nM rICOSL.Fcvariant proteins. This mixture was added to tissue culture treated flatbottom 96 well plates (Corning, USA) overnight to facilitate adherenceof the stimulatory proteins to the wells of the plate. The next day,unbound protein was washed off the plates and 100,000 purified human panT cells (BenTech Bio, US) or human T cell clone BC3 (Astarte Biologics,USA) were added to each well in a final volume of 200 μl of Ex-Vivo 15media (Lonza, Switzerland). In some instances, human pan T cells werelabeled with 0.25 uM carboxyfluorescein succinimidyl ester (CFSE,ThermoFisher Scientific, USA). Cells were cultured 3 days beforeharvesting culture supernatants and measuring human IFN-gamma levelswith Duoset ELISA kit (R&D Systems, USA) as mentioned above. Cellularproliferation was determined by the percent of input cells that entereddivision as measured by CFSE dilution on cells stained withfluorescently-conjugated anti-CD4, anti-CD8 antibodies (BD, USA) ortotal T cells via flow cytometric analysis on an LSR II (BD, USA),

C. Results

Results for the binding and activity studies for exemplary testedvariants are shown in Table 7 which indicates exemplary IgSF domainamino acid substitutions (replacements) in the ECD of ICOSL selected inthe screen for affinity-maturation against the respective cognatestructures ICOS and CD28. In the Tables, the exemplary amino acidsubstitutions are designated by amino acid position number correspondingto the respective reference (e.g., unmodified) ECD sequence as follows.For example, the reference (e.g., unmodified) ECD sequence in Table 7(WT ICOSL) is the (e.g., unmodified) ICOSL ECD sequence set forth in SEQID NO: 32. The amino acid position is indicated in the middle, with thecorresponding reference (e.g. unmodified or wild-type) amino acid listedbefore the number and the identified variant amino acid substitutionlisted after the number. Column 2 sets forth the SEQ ID NO identifierfor the variant ECD for each variant ECD-Fc fusion molecule.

Also shown is the binding activity as measured by the Mean FluorescenceIntensity (MFI) value for binding of each variant Fc-fusion molecule tocells transfected to express the cognate ligand and the ratio of the MFIcompared to the binding of the corresponding reference (e.g.,unmodified) ECD-Fc fusion molecule not containing the amino acidsubstitution(s) to the same cell-expressed counter structure ligand. Thefunctional activity of the variant Fc-fusion molecules to modulate theactivity of T cells also is shown based on the calculated levels ofIFN-gamma in culture supernatants (pg/mL) generated either i) with theindicated variant ECD-Fc fusion molecule coimmoblized with anti-CD3 orii) with the indicated variant ECD-Fc fusion molecule in an MLR assay.The Table also depicts the ratio of IFN-gamma produced by each variantECD-Fc compared to the corresponding reference (e.g., unmodified orwild-type) ECD-Fc in both functional assays.

As shown, the selections resulted in the identification of a number ofICOSL IgSF domain variants that were affinity-modified to exhibitincreased binding for at least one, and in some cases more than one,cognate counter structure ligand. In addition, the results showed thataffinity modification of the variant molecules also exhibited improvedactivities to both increase and/or decrease immunological activitydepending on the format of the molecule. For example, coimmobilizationof the ligand likely provides a multivalent interaction with the cell tocluster or increase the avidity to favor agonist activity and increase Tcell activation compared to the reference (e.g. unmodified or wildtype)ECD-Fc molecule not containing the amino acid replacement(s). However,when the molecule is provided as a bivalent Fc molecule in solution, thesame IgSF domain variants exhibited an antagonist activity to decrease Tcell activation compared to the reference (e.g. unmodified or wildtype)ECD-Fv molecule not containing the amino acid replacement(s).

TABLE 7 ICOSL variants selected against CD28 or ICOS. Moleculesequences, binding data, and costimulatory bioactivity data.Coimmobilization with anti-CD3 MLR Binding IFN-gamma IFN-gamma SEQ ICOSOD CD28 MFI pg/mL levels pg/mL ID NO (parental (parental (parental(parental ICOSL mutation(s) (ECD) ratio) ratio) ratio) ratio) N52S 1091.33 162 1334    300 (1.55) (9.00) (1.93)  (0.44) N52H 110 1.30 3681268    39 (1.51) (20.44) (1.83)  (0.06) N52D 111 1.59 130 1943    190(1.85) (7.22) (2.80)  (0.28) N52Y/N57Y/ 112 1.02 398 510*    18F138L/L203P (1.19) (22.11) (1.47*) (0.03) N52H/N57Y/Q100P 113 1.57 4472199    25 (1.83) (24.83) (3.18)  (0.04) N52S/Y146C/Y152C 114 1.26 391647    152 (1.47) (2.17) (2.38)  (0.22) N52H/C198R 115 1.16 363 744*   ND (1.35) (20.17) (2.15*) (ND) N52H/C140del/ 372 ND 154 522*    ND T225A(ND) (8.56) (1.51*) (ND) N52H/C198R/T225A 117 1.41 344 778*    0 (1.64)(19.11) (2.25*) (0) N52H/K92R 118 1.48 347 288*    89 (1.72) (19.28)(0.83*) (0.13) N52H/S99G 119 0.09 29 184*    421 (0.10) (1.61) (0.53*)(0.61) N52Y 120 0.08 18 184*    568 (0.09) (1.00) (0.53*) (0.83) N57Y121 1.40 101 580*    176 (1.63) (5.61) (1.68*) (0.26) N57Y/Q100P 1220.62 285 301*    177 (0.72) (15.83) (0.87*) (0.26) N52S/S130G/Y152C 1230.16 24 266*    1617 (0.19) (1.33) (0.77*) (2.35) N52S/Y152C 124 0.18 29238*    363 (0.21) (1.61) (0.69*) (0.53) N52S/C198R 125 1.80 82 1427   201 (2.09) (4.56) (2.06)  (0.29) N52Y/N57Y/Y152C 126 0.08 56 377*    439(0.09) (3.11) (1.09*) (0.64) N52Y/N57Y/ 127 ND 449 1192    NDH129P/C198R (ND) (24.94) (1.72)  (ND) N52H/L161P/C198R 128 0.18 343643*    447 (0.21) (19.05) (1.86*) (0.65) N52S/T113E 129 1.51 54 451*   345 (1.76) (3.00) (1.30*) (0.50) S54A 130 1.62 48 386*    771 (1.88)(2.67) (1.12*) (1.12) N52D/S54P 368 1.50 38 476*    227 (1.74) (2.11)(1.38*) (0.33) S54F/V193A 905 0.51 16 294    262 (0.59) (0.87) (0.85) (0.38) N52K/L208P 132 1.91 291 1509    137 (2.22) (16.17) (2.18)  (0.20)N52S/Y152H 133 0.85 68 2158    221 (0.99) (3.78) (3.12)  (0.32)N52D/V151A 134 0.90 19 341*    450 (1.05) (1.06) (0.99*) (0.66)N52H/I143T 135 1.83 350 2216    112 (2.13) (19.44) (3.20)  (0.16)N52S/L80P 136 0.09 22 192*    340 (0.10) (1.22) (0.55*) (0.49)F120S/Y152H/N201S 137 0.63 16 351*    712 (0.73) (0.89) (1.01*) (1.04)N52S/R75Q/L203P 138 1.71 12 1996    136 (1.99) (0.67) (2.88)  (0.20)N52S/D158G 139 1.33 39 325*    277 (1.55) (2.17) (0.94*) (0.40)N52D/Q133H 140 1.53 104 365*    178 (1.78) (5.78) (1.05*) (0.26) WTICOSL 32 0.86 18 692/346* 687 (1.00) (1.00) (1.00)  (1.00) *Parentalratio calculated using 346 pg/mL IFN-gamma for WT ICOSL

Binding assays were repeated substantially as described above, exceptthat binding also was assessed against cells expressing full-lengthhuman CTLA4. ICOSL variant Fc fusion proteins also were further assessedin an anti-CD3 coimmobilization assay substantially as described above.The results confirmed identification of a number of ICOSL IgSF domainvariants that exhibited increased binding affinity for at least one, andin some cases more than one, cognate ligand. In addition, the resultsshowed that affinity modification of the variant molecules alsoexhibited improved activities in the coimmobilization assay.

Example 7 Additional Affinity Modified IgSF Domains

This examples describe the design, creation, and screening of additionalaffinity modified CD80 (B7-1), CD86 (B7-2) and NKp30 immunomodulatoryproteins, which are other components of the immune synapse (IS) thathave a demonstrated dual role in both immune activation and inhibition.These examples demonstrate that affinity modification of IgSF domainsyields proteins that can act to both increase and decrease immunologicalactivity. This work also describes the various combinations of thosedomains fused in pairs (i.e., stacked) with a variant affinity modifiedICOSL to form a Type II immunomodulatory protein to achieveimmunomodulatory activity.

Mutant DNA constructs of human CD80, CD86 and NKp30 IgSF domains fortranslation and expression as yeast display libraries were generatedsubstantially as described in Example 1. For libraries that targetspecific residues of target protein for complete or partialrandomization with degenerate codons, the coding DNA's for theextracellular domains (ECD) of human CD80 (SEQ ID NO:28), and NKp30 (SEQID NO:54) were ordered from Integrated DNA Technologies (Coralville,Iowa) as a set of overlapping oligonucleotides of up to 80 base pairs(bp) in length. Alternatively, residues were mutated by site-directedtargeted mutagenesis substantially as described in Example 1.Alternatively, random libraries were constructed to identify variants ofthe ECD of CD80 (SEQ ID NO:28), CD86 (SEQ ID NO: 29) and NKp30 (SEQ IDNO:54) substantially as described in Example 1.

The targeted and random library DNA was introduced into yeastsubstantially as described in Example 2 to generate yeast libraries. Thelibraries were used to select yeast expressing affinity modifiedvariants of CD80, CD86 and NKp30 substantially as described in Example3. Cells were processed to reduce non-binders and to enrich for CD80,CD86 or NKp30 variants with the ability to bind their exogenousrecombinant counter-structure proteins substantially as described inExample 3. For example, yeast displayed targeted or random CD80libraries were selected against each of CD28, CTL-4, and PD-L1,separately. This was then followed by two to three rounds of flowcytometry sorting using exogenous counter-structure protein staining toenrich the fraction of yeast cells that displays improved binders.Magnetic bead enrichment and selections by flow cytometry areessentially as described in Keith D. Miller, 1 Noah B. Pefaur, 2 andCheryl L. Baird1 Current Protocols in Cytometry 4.7.1-4.7.30, July 2008.

With CD80, CD86 and NKp30 libraries, target ligand proteins were sourcedfrom R&D Systems (USA) as follows: human rCD28.Fc (i.e., recombinantCD28-Fc fusion protein), rPDL1.Fc, rCTLA4.Fc, and rB7H6.Fc. Two-colorflow cytometry was performed substantially as described in Example 3.Yeast outputs from the flow cytometric sorts were assayed for higherspecific binding affinity. Sort output yeast were expanded andre-induced to express the particular IgSF affinity modified domainvariants they encode. This population then can be compared to theparental, wild-type yeast strain, or any other selected outputs, such asthe bead output yeast population, by flow cytometry.

In the case of NKp30 yeast variants selected for binding to B7-H6, theF2 sort outputs gave MFI values of 533 when stained with 16.6 nMrB7H6.Fc, whereas the parental NKp30 strain MFI was measured at 90 whenstained with the same concentration of rB7H6.Fc (6-fold improvement).

Among the NKp30 variants that were identified, was a variant thatcontained mutations L30V/A60V/S64P/S86G with reference to positions inthe NKp30 extracellular domain corresponding to positions set forth inSEQ ID NO:54. Among the CD86 variants that were identified, was avariant that contained mutations Q35H/H90L/Q102H with reference topositions in the CD86 extracellular domain corresponding to positionsset forth in SEQ ID NO: 29. Among the CD80 variants that wereidentified, were variants set forth in Table 8 and described furtherbelow.

As with ICOSL, the MFIs of all F2 outputs described above when measuredwith the anti-HA tag antibody on FL1 did not increase and sometimes wentdown compared to wild-type strains, indicating that increased bindingwas not a function of increased expression of the selected variants onthe surface of yeast, and validated gating strategies of only selectingmid to low expressors with high ligand binding.

Exemplary selection outputs were reformatted as immunomodulatoryproteins containing an affinity modified (variant) extracellular domain(ECD) of CD80 fused to an Fc molecule (variant ECD-Fc fusion molecules)substantially as described in Example 4 and the Fc-fusion protein wasexpressed and purified substantially as described in Example 5.

Binding of exemplary CD80 Fc-fusion variants to cell-expressed counterstructures was then assessed substantially as described in Example 6. Toproduce cells expressing cognate binding partners, full-length mammaliansurface expression constructs for each of human CD28, CTLA4 and PD-L1were produced substantially as described in Example 6. Binding studiesand flow cytometry were carried out substantially as described inExample 6. In addition, the bioactivity of the Fc-fusion variant proteinwas characterized by either mixed lymphocyte reaction (MLR) or anti-CD3coimmobilization assay substantially as described in Example 6.

Results for the binding and activity studies for exemplary testedvariants are shown in Tables 8 and 9. In particular, Table 8 indicatesexemplary IgSF domain amino acid substitutions (replacements) in the ECDof CD80 selected in the screen for affinity-maturation against therespective cognate structure CD28. Table 9 indicates exemplary IgSFdomain amino acid substitutions (replacements) in the ECD of CD80selected in the screen for affinity-maturation against the respectivecognate structure PD-L1. As above, for each Table, the exemplary aminoacid substitutions are designated by amino acid position numbercorresponding to the respective reference (e.g., unmodified) ECDsequence as follows. For example, the reference (e.g., unmodified) ECDsequence in Tables 8 and 9 is the unmodified CD80 ECD sequence set forthin SEQ ID NO:28. The amino acid position is indicated in the middle,with the corresponding reference (e.g., unmodified) amino acid listedbefore the number and the identified variant amino acid substitutionlisted after the number. Column 2 sets forth the SEQ ID NO identifierfor the variant ECD for each variant ECD-Fc fusion molecule.

Also shown is the binding activity as measured by the Mean FluorescenceIntensity (MFI) value for binding of each variant Fc-fusion molecule tocells engineered to express the cognate counter structure ligand and theratio of the MFI compared to the binding of the corresponding reference(e.g., unmodified) ECD-Fc fusion molecule not containing the amino acidsubstitution(s) to the same cell-expressed counter structure ligand. Thefunctional activity of the variant Fc-fusion molecules to modulate theactivity of T cells also is shown based on the calculated levels ofIFN-gamma in culture supernatants (pg/mL) generated either i) with theindicated variant ECD-Fc fusion molecule coimmoblized with anti-CD3 orii) with the indicated variant ECD-Fc fusion molecule in an MLR assay.The Tables also depict the ratio of IFN-gamma produced by each variantECD-Fc compared to the corresponding reference (e.g., unmodified) ECD-Fcin both functional assays.

As shown, the selections resulted in the identification of a number ofCD80 IgSF domain variants that were affinity-modified to exhibitincreased binding for at least one, and in some cases more than one,cognate counter structure ligand. In addition, the results showed thataffinity modification of the variant molecules also exhibited improvedactivities to both increase and decrease immunological activitydepending on the format of the molecule. For example, coimmobilizationof the ligand likely provides a multivalent interaction with the cell tocluster or increase the avidity to favor agonist activity and increase Tcell activation compared to the reference (e.g. unmodified or wildtype)ECD-Fc molecule not containing the amino acid replacement(s). However,when the molecule is provided as a bivalent Fc molecule in solution, thesame IgSF domain variants exhibited an antagonist activity to decrease Tcell activation compared to the reference (e.g. unmodified or wildtype)ECD-Fc molecule not containing the amino acid replacement(s).

TABLE 8 CD80 variants selected against CD28. Molecule sequences, bindingdata, and costimulatory bioactivity data. Binding Coimmobilization MLRwith anti-CD3 IFN-gamma CD28 CTLA-4 PD-L1 IFN-gamma levels SEQ MFI MFIMFI pg/mL pg/mL ID NO (parental (parental (parental (parental (parentalCD80 mutation(s) (ECD) ratio) ratio) ratio) ratio) ratio)L70Q/A91G/N144D 508 125 (1.31) 283 (1.36) 6 (0.08) 93 (1.12) 716 (0.83)L70Q/A91G/T130A 56 96 (1.01) 234 (1.13) 7 (0.10) 99 (1.19) 752 (0.87)L70Q/A91G/I118A/T120S/T130A/K169E 59 123 (1.29) 226 (1.09) 7 (0.10) 86(1.03) 741 (0.86) V4M/L70Q/A91G/I118V/T20S/T130A/ 510 89 (0.94) 263(1.26) 6 (0.09) 139 (1.67) 991 (1.14) K169EL70Q/A91G/I118V/T120S/T130A/K169E 59 106 (1.12) 263 (1.26) 6 (0.09) 104(1.25) 741 (0.86) V20L/L70Q/A91S/I118V/T120S/T130A 513 105 (1.11) 200(0.96) 9 (0.13) 195 (2.34) 710 (0.82) S44P/L70Q/A91G/T130A 61 88 (0.92)134 (0.64) 5 (0.07) 142 (1.71) 854 (0.99)L70Q/A91G/E117G/I118V/T120S/T130A 514 120 (1.27) 193 (0.93) 6 (0.08) 98(1.05) 736 (0.85) A91G/I118V/T120S/T130A 515 84 (0.89) 231 (1.11) 44(0.62) 276 (3.33) 714 (0.82) L70R/A91G/I118V/T120S/T130A/T199S 516 125(1.32) 227 (1.09) 6 (0.09) 105 (1.26) 702 (0.81)L70Q/E81A/A91G/I118V/T120S/1127T/ 517 140 (1.48) 185 (0.89) 18 (0.25) 98(1.18) 772 (0.89) T130A L70Q/Y87N/A91G/T130A 66 108 (1.13) 181 (0.87) 6(0.08) 136 (1.63) 769 (0.89) T28S/L70Q/A91G/I118V/E95K/T120S/ 518 32(0.34) 65 (0.31) 6 (0.08) 120 (1.44) 834 (0.96) I126V/T130A/K169EN63S/L70Q/A91G/S114T/I118V/ 519 124 (1.30) 165 (0.79) 6 (0.08) 116(1.39) 705 (0.81) T120S/T130A K36E/I67T/L70Q/A91G/I118V/ 520 8 (0.09) 21(0.10) 5 (0.08) 53 (0.63) 852 (0.98) T120S/T130A/N152TE52G/L70Q/A91G/D107N/I118V/ 521 113 (1.19) 245 (1.18) 6 (0.08) 94 (1.13)874 (1.01) T120S/T130A K169E K37E/F59S/L70Q/A91G/I118V/T120S/ 522 20(0.21) 74 (0.36) 6 (0.08) 109 (1.31) 863 (1.00) T130A/K185E A91G/S103P72 39 (0.41) 56 (0.27) 9 (0.13) 124 (1.49) 670 (0.77) K89E/T130A 73 90(0.95) 148 (0.71) 75 (1.07) 204 (2.45) 761 (0.88) A91G 74 96 (1.01) 200(0.96) 85 (1.21) 220 (2.65) 877 (1.01) D60V/A91G/I118V/T120S/T130A/K169E523 111 (1.17) 222 (1.07) 12 (0.18) 120 (1.44) 744 (0.86)K54M/L70Q/A91G/Y164H 524 68 (0.71) 131 (0.63) 5 (0.08) 152 (1.83) 685(0.79) M38T/L70Q/E77G/A91G/I118V/T120S/ 525 61 (0.64) 102 (0.49) 5(0.07) 119 (1.43) 796 (0.92) T130A/N152T R29H/E52G/L70R/E88G/A91G/T130A78 100 (1.05) 119 (0.57) 5 (0.08) 200 (2.41) 740 (0.85)Y31H/T41G/M43L/L70Q/A91G/ 526 85 (0.89) 85 (0.41) 6 (0.08) 288 (3.47)782 (0.90) I118V/T120S/I126V/T130A V68A/T110A 80 103 (1.08) 233 (1.12)48 (0.68) 163 (1.96) 861 (0.99) L65H/D90G/T110A/F116L 527 33 (0.35) 121(0.58) 11 (0.15) 129 (1.55) 758 (0.88) R29H/E52G/D90N/I118V/T120S/T130A82 66 (0.69) 141 (0.68) 11 (0.15) 124 (1.49) 800 (0.92) A91G/L102S 83 6(0.06) 6 (0.03) 5 (0.08) 75 (0.90) 698 (0.81) I67T/L70Q/A91G/I118V T120S530 98 (1.03) 160 (0.77) 5 (0.08) 1751 (21.1) 794 (0.92)L70Q/A91G/T110A/I118V/T120S/T130A 531 8 (0.09) 14 (0.07) 5 (0.07) 77(0.93) 656 (0.76) M38V/T41D/M43I/W50G/D76G/V83A/ 532 5 (0.06) 8 (0.04) 8(0.11) 82 (0.99) 671 (0.78) K89E/I118V/T120S/I126V/T130A V22A/L70Q/S121P87 5 (0.06) 7 (0.04) 5 (0.07) 105 (1.27) 976 (1.13)A12V/S15F/Y31H/M38L/T41G/M43L/ 533 6 (0.06) 6 (0.03) 5 (0.08) 104 (1.25)711 (0.82) D90N/T130A/P137L/N149D N152T I67F/L70R/E88G/A91G/I118V/T120S/534 5 (0.05) 6 (0.03) 6 (0.08) 62 (0.74) 1003 (1.16) T130AE24G/L25P/L70Q/A91G/I118V/T120S/ 535 26 (0.27) 38 (0.18) 8 (0.11) 101(1.21) 969 (1.12) N152T A91G/F92L/F108L/I118V/T120S 536 50 (0.53) 128(0.61) 16 (0.11) 59 (0.71) 665 (0.77) WT CD80 28 95 (1.00) 208 (1.00) 70(1.00) 83 (1.00) 866 (1.00)

TABLE 9 CD80 variants selected against PD-L1. Molecule sequences,binding data, and costimulatory bioactivity data. BindingCoimmobilization MLR with anti-CD3 IFN-gamma CD28 CTLA-4 PD-L1 IFN-gammalevels SEQ MFI MFI MFI Pg/mL Pg/mL ID NO (parental (parental (parental(parental (parental CD80 mutation(s) (ECD) ratio) ratio) ratio) ratio)ratio) R29D/Y31L/Q33H/ 92 1071 (0.08) 1089 (0.02) 37245 (2.09) 387(0.76) 5028 (0.26) K36G/M38I/T41A/ M43R/M47T/E81V/ L85R/K89N/A91T/F92P/K93V/ R94L/ I118T/N149S R29D/Y31L/Q33H/ 93 1065 (0.08) 956 (0.02)30713 (1.72) 400 (0.79) 7943 (0.41) K36G/M38I/T41A/ M43R/M47T/E81V/L85R/K89N/A91T/ F92P/K93V/R94L/ N144S/N149S R29D/Y31L/Q33H/ 94 926(0.07) 954 (0.02) 47072 (2.64) 464 (0.91) 17387 (0.91) K36G/M38I/T41A/M42T/M43R/M47T/ E81V/L85R/K89N7 A91T/F92P/K93V/ R94L/L148S/N149SE24G/R29D/Y31L/ 95 1074 (0.08) 1022 (0.02) 1121 (0.06) 406 (0.80) 13146(0.69) Q33H/K36G/M38I/ T41A/M43R/M47T/ F59L/E81V/L85R/ K89N/A91T/F92P/K93V/R94L/H96R R29D/Y31L/Q33H/ 96 1018 (0.08) 974 (0.02) 25434 (1.43)405 (0.80) 24029 (1.25) K36G/M38I/T41A/ M43R/M47T/E81V/ L85R/K89N/A91T/F92P/K93V/R94L/N149S R29V/M43Q/E81R/ 97 1029 (0.08) 996 (0.02) 1575(0.09) 342 (0.67) 11695 (0.61) L85I/K89R/D90L/ A91E/F92N/K93Q/R94GT41I/A91G 98 17890 (1.35) 50624 (1.01) 12562 (0.70) 433 (0.85) 26052(1.36) E88D/K89R/D90K/A91G/ 537 41687 (3.15) 49429 (0.99) 20140 (1.13)773 (1.52) 6345 (0.33) F92Y/K93R/N122S/ N178S E88D/K89R/D90K/A91G/ 53851663 (3.91) 72214 (1.44) 26405 (1.48) 1125 (2.21) 9356 (0.49) F92Y/K93RK36G/K37Q/M38I/ 539 1298 (0.10) 1271 (0.03) 3126 (0.18) 507 (1.00) 3095(0.16) L40M/F59L/E81V/L85R/ K89N/A91T/F92P/ K93V/R94L/E99G/ T130A/N149SAE88D/K89R/D90K/ 102 31535 (2.38) 50868 (1.02) 29077 (1.63) 944 (1.85)5922 (0.31) A91G/F92Y/K93R K36G/K37Q/M38I/L40M 103 1170 (0.09) 1405(0.03) 959 (0.05) 427 (0.84) 811 (0.04) K36G/L40M 540 29766 (2.25) 58889(1.18) 20143 (1.13) 699 (1.37) 30558 (1.59) WTCD80 28 13224 (1.00) 50101(1.00) 17846 (1.00) 509 (1.00) 19211 (1.00)

Generation and Assessment of Stacked Molecules Containing DifferentAffinity-Modified Domains

This Example describes further immunomodulatory proteins that weregenerated as stack constructs containing at least two different affinitymodified domains from identified variant ICOSL polypeptides and one moreadditional variant CD80, CD86, ICOSL, and NKp30 molecules linkedtogether and fused to an Fc.

Selected variant molecules described above that were affinity-modifiedfor one or more counter structure ligand were used to generate “stack”molecule (i.e., Type II immunomodulatory protein) containing two or moreaffinity-modified IgSF domains. Stack constructs were obtained asgeneblocks (Integrated DNA Technologies, Coralville, Iowa) that encodethe stack in a format that enables its fusion to Fc by standard Gibsonassembly using a Gibson assembly kit (New England Biolabs, USA).

The encoding nucleic acid molecule of all stacks was generated to encodea protein designed as follows: Signal peptide, followed by the firstvariant IgV of interest, followed by a 15 amino acid linker which iscomposed of three GGGGS(G4S) motifs (SEQ ID NO:228), followed by thesecond IgV of interest, followed by two GGGGS linkers (SEQ ID NO: 229)followed by three alanines (AAA), followed by a human IgG1 Fc asdescribed above. To maximize the chance for correct folding of the IgVdomains in each stack, the first IgV was preceded by all residues thatnormally occur in the wild-type protein between this IgV and the signalpeptide (leading sequence). Similarly, the first IgV was followed by allresidues that normally connect it in the wild-type protein to either thenext Ig domain (typically an IgC domain) or if such a second IgV domainis absent, the residues that connect it to the transmembrane domain(trailing sequence). The same design principle was applied to the secondIgV domain except that when both IgV domains were derived from sameparental protein (e.g. a ICOSL IgV stacked with another ICOSL IgV), thelinker between both was not duplicated.

Table 10 sets forth the design for exemplary stacked constructs. Theexemplary stack molecules shown in Table 10 contains the Ig domains(e.g. IgV domain) as indicated and additionally trailing sequences asdescribed above. In the Table, the following components are present inorder: signal peptide (SP; SEQ ID NO:225), Ig domain 1 (e.g. Ig1),trailing sequence 1 (TS1), linker 1 (LR1; SEQ ID NO:228), Ig domain 2(Ig2), trailing sequence 2 (TS2), linker 2 (LR2; SEQ ID NO:230) and Fcdomain (SEQ ID NO:226 containing C5S/R77C/N82G/V87C amino acidsubstitution). In some cases, a leading sequence 1(LS1) is presentbetween the signal peptide and IgV1 and in some cases a leading sequence2 (LS2) is present between the linker and IgV2.

TABLE 10 Amino acid sequence (SEQ ID NO) of components of exemplarystacked constructs First domain Second domain SP LS1 Ig1 TS1 LR1 LS2 Ig2TS2 LR2 Fc Domain 1: NKp30 + — 214 235 + — 196 233 + + WT Domain 2:ICOSL WT Domain 1: NKp30 + — 215 235 + — 212 233 + + L30V/A60V/S64P/S86G Domain 2: ICOSL N52S/N57Y/H94D/ L96F/L98F/Q100R Domain 1: NKp30 + —215 235 + — 199 233 + + L30V/A60V/S64P/ S86G) Domain 2: ICOSL N52DDomain 1: NKp30 + — 215 235 + — 201 233 + + L30V/A60V/S64P/ S86G Domain2: ICOSL N52H/N57Y/Q100P Domain 1: ICOSL + — 196 233 + — 214 235 + + WTDomain 2: Nkp30 WT Domain 1: ICOSL + — 199 233 + — 215 235 + + N52DDomain 2: NKp30 L30V/A60V/S64P/ S86G Domain 1: ICOSL + — 201 233 + — 215235 + + N52H/N57Y/Q100P Domain 2: NKp30 L30V/A60V/S64P/ S86G Domain 1:CD80 + — 152 471 + — 196 233 + + WT Domain 2: ICOSL WT Domain 1: CD80 +— 189 471 + — 213 233 + + E88D/K89R/D90K/ A91G/F92Y/K93R Domain 2: ICOSLN52S/N57Y/H94D/ L96F/L98F/Q100R/ G103E/F120S Domain 1: CD80 + — 193471 + — 213 233 + + A12T/H18L/M43V/ F59L/E77K/P109S/ I118T Domain 2:ICOSL N52S/N57Y/H94D/ L96F/L98F/Q100R/ G103E/F120S Domain 1: CD80 + —193 471 + — 199 233 + + A12T/H18L/M43V/ F59L/E77K/P109S/ I118T Domain 2:ICOSL N52D Domain 1: CD80 + — 189 471 + — 201 233 + + E88D/K89R/D90K/A91G/F92Y/K93R Domain 2: ICOSL N52H/N57Y/Q100P Domain 1: CD80 + — 193471 + — 201 233 + + A12T/H18L/M43V/ F59L/E77K/P109S/ I118T Domain 2:ICOSL N52H/N57Y/Q100P Domain 1: ICOSL + — 196 233 + — 152 471 + + WTDomain 2: CD80 WT Domain 1: ICOSL + — 213 233 + — 189 471 + +N52S/N57Y/H94D/ L96F/L98F/Q100R/ G103E/F120S Domain 2: CD80E88D/K89R/D90K/ A91G/F92Y/K93R Domain 1: ICOSL + — 213 233 + — 193471 + + N52S/N57Y/H94D/ L96F/L98F/Q100R/ G103E/F120S Domain 2: CD80A12T/H18L/M43V/ F59L/E77K/P109S/ I118T Domain 1: ICOSL + — 199 233 + —189 471 + + N52D Domain 2: CD80 E88D/K89R/D90K/ A91G/F92Y/K93R Domain 1:ICOSL + — 199 233 + — 193 471 + + N52D Domain 2: CD80 A12T/H18L/M43V/F59L/E77K/P109S/ I118T Domain 1: ICOSL + — 201 233 + — 189 471 + +N52H/N57Y/Q100P Domain 2: CD80 E88D/K89R/D90K/ A91G/F92Y/K93R Domain 1:ICOSL + — 201 233 + — 193 471 + + N52H/N57Y/Q100P Domain 2: CD80A12T/H18L/M43V/ F59L/E77K/P109S/ I118T Domain 1: CD86 + 236 220 237 + —196 233 + + WT Domain 2: ICOSL WT Domain 1: CD80 + — 192 471 + — 213233 + + R29H/Y31H/T41G/ Y87N/E88G/K89E/ D90N/A91G/P109S Domain 2: ICOSLN52S/N57Y/H94D/ L96F/L98F/Q100R/ G103E/F120S Domain 1: CD80 + — 175471 + — 213 233 + + I67T/L70Q/A91G/ T120S Domain 2: ICOSLN52S/N57Y/H94D/ L96F/L98F/Q100R/ G103E/F120S Domain 1: CD80 + — 192471 + — 199 233 + + R29H/Y31H/T41G/ Y87N/E88G/K89E/ D90N/A91G/P109SDomain 2: ICOSL N52D Domain 1: CD80 + — 175 471 + — 199 233 + +I67T/L70Q/A91G/ T120S Domain 2: ICOSL N52D Domain 1: CD80 + — 192 471 +— 201 233 + + R29H/Y31H/T41G/ Y87N/E88G/K89E/ D90N/A91G/P109S Domain 2:ICOSL N52H/N57Y/Q100P Domain 1: CD80 + — 175 471 + — 201 233 + +I67T/L70Q/A91G/ T120S Domain 2: ICOSL N52H/N57Y/Q100P Domain 1: CD86 +236 221 237 + — 213 233 + + Q35H/H90L/Q102H Domain 2: ICOSLN52S/N57Y/H94D/ L96F/L98F/Q100R/ G103E/F120S Domain 1: CD86 + 236 221237 + — 199 233 + + Q35H/H90L/Q102H Domain 2: ICOSL N52D Domain 1:CD86 + 236 221 237 + — 201 233 + + Q35H/H90L/Q102H Domain 2: ICOSLN52H/N57Y/Q100P Domain 1: ICOSL + — 196 233 + 236 220 237 + + WT Domain2: CD86 WT Domain 1: ICOSL + — 213 233 + — 192 471 + + N52S/N57Y/H94D/L96F/L98F/Q100R/ G103E/F120S Domain 2: CD80 R29H/Y31H/T41G/Y87N/E88G/K89E/ D90N/A91G/P109S Domain 1: ICOSL + — 213 233 + — 175471 + + N52S/N57Y/H94D/ L96F/L98F/Q100R/ G103E/F120S Domain 2: CD80I67T/L70Q/A91G/ T120S Domain 1: ICOSL + — 199 233 + — 192 471 + + N52DDomain 2: CD80 R29H/Y31H/T41G/ Y87N/E88G/K89E/ D90N/A91G/P109S Domain 1:ICOSL + — 199 233 + — 175 471 + + N52D Domain 2: CD80 I67T/L70Q/A91G/T120S Domain 1: ICOSL + — 201 233 + — 192 471 + + N52H/N57Y/Q100P Domain2: CD80 R29H/Y31H/T41G/ Y87N/E88G/K89E/ D90N/A91G/P109S Domain 1:ICOSL + — 213 233 + 236 221 237 + + N52S/N57Y/H94D/ L96F/L98F/Q100R/G103E/F120S Domain 2: CD86 Q35H/H90L/Q102H Domain 1: ICOSL + — 199 233 +236 221 237 + + N52D Domain 2: CD86 Q35H/H90L/Q102H Domain 1: ICOSL + —201 233 + 236 221 237 + + N52H/N57Y/Q100P Domain 2: CD86 Q35H/H90L/Q102H

High throughput expression and purification of the variantIgV-stacked-Fc fusion molecules containing various combinations ofvariant IgV domains from CD80, CD86, ICOSL or Nkp30 containing at leastone affinity-modified IgV domain were generated substantially asdescribed in Example 5. Binding of the variant IgV-stacked-Fc fusionmolecules to respective counter structures and functional activity byanti-CD3 coimmobilization assay also were assessed substantially asdescribed in Example 6. For example, costimulatory bioactivity of thestacked IgSF Fc fusion proteins was determined in a similar immobilizedanti-CD3 assay as above. In this case, 4 nM of anti-CD3 (OKT3,Biolegend, USA) was coimmobilized with 4 nM to 120 nM of human rB7-H6.Fc(R&D Systems, USA) or human rPD-L1.Fc (R&D Systems, USA) overnight ontissue-culture treated 96-well plates (Corning, USA). The following dayunbound protein was washed off with PBS and 100,000 purified pan T cellswere added to each well in 100 μl Ex-Vivo 15 media (Lonza, Switzerland).The stacked IgSF domains were subsequently added at concentrationsranging from 8 nM to 40 nM in a volume of 100 al for 200 al volumetotal. Cells were cultured 3 days before harvesting culture supernatantsand measuring human IFN-gamma levels with Duoset ELISA kit (R&D Systems,USA) as mentioned above.

The results are set forth in Tables 11-13. Specifically, Table 11 setsforth binding and functional activity results for variant IgV-stacked-Fcfusion molecules containing an NKp30 IgV domain and an ICOSL IgV domain.Table 12 and 13 sets forth binding and functional activity results forvariant IgV-stacked-Fc fusion molecules containing a variant ICOSL IgVdomain and a variant CD80 IgV or CD86 IgV domain.

For each of Tables 11-13, Column 1 indicates the structural organizationand orientation of the stacked, affinity modified or wild-type (WT)domains beginning with the amino terminal (N terminal) domain, followedby the middle WT or affinity modified domain located before the Cterminal human IgG1 Fc domains. Column 2 sets forth the SEQ ID NOidentifier for the sequence of each IgV domain contained in a respective“stack” molecule. Column 3 shows the binding partners which theindicated affinity modified stacked domains from column 1 were selectedagainst.

Also shown is the binding activity as measured by the Mean FluorescenceIntensity (MFI) value for binding of each stack molecule to cellstransfected to express various counter structure ligands and the ratioof the MFI compared to the binding of the corresponding stack moleculecontaining reference (e.g., unmodified) IgV domains not containing theamino acid substitution(s) to the same cell-expressed counter structureligand. The functional activity of the variant stack molecules tomodulate the activity of T cells also is shown based on the calculatedlevels of IFN-gamma in culture supernatants (pg/mL) generated with theindicated variant stack molecule in solution and the appropriate ligandcoimmoblized with anti-CD3 as described in Example 6. The Table alsodepicts the ratio of IFN-gamma produced by each variant stack moleculecompared to the corresponding reference (e.g., unmodified) stackmolecule in the coimmobilization assay.

As shown, the results showed that it was possible to generate stackmolecules containing at least one variant IgSF domains that exhibitedaffinity-modified activity of increased binding for at least one cognatecounter structure ligand compared to a corresponding stack moleculecontaining the respective reference (e.g. wild-type or unmodified) IgVdomain. In some cases, the stack molecule, either from one or acombination of both variant IgSF domains in the molecule, exhibitedincreased binding for more than one cognate counter structure ligand.The results also showed that the order of the IgV domains in the stackedmolecules could, in some cases, alter the degree of improved bindingactivity. In some cases, functional T cell activity also was alteredwhen assessed in the targeted coimmobilization assay.

TABLE 11 Stacked variant IgV Fc fusion proteins containing an NKp30 IgVdomain and an ICOSL IgV domain Anti-CD3 Domain Structure SEQ CounterBinding Activity coimmobilization N terminal to C ID NO structure B7H6MFI ICOS MFI CD28 MFI assay pg/mL IFN- terminal: domain (Ig selected (WTparental (WT parental (WT parental gamma (WT parental 1/domain 2/Fcdomain) against MFI ratio) MFI ratio) MFI ratio) IFN-gamma ratio) Domain1: NKp30 WT 214 — 64538 (1.00) 26235 (1.00) 6337 (1.00) 235 (1.00)Domain 2: ICOSL WT 196 Domain 1: NKp30 215 B7-H6 59684 (0.92) 12762(0.49) 9775 (1.54) 214 (0.91) L30V/A60V/S64P/S86G Domain 2: ICOSL 212ICOS-CD28 N52S N57Y H94D L96F L98F Q100R Domain 1: NKp30 215 B7-H6 65470(1.01) 30272 (1.15) 9505 (1.50) 219 (0.93) L30V/A60V/S64P/S86G Domain 2:ICOSL 199 ICOS-CD28 N52D Domain 1: NKp30 215 B7-H6 38153 (0.59) 27903(1.06) 11300 (1.78) 189 (0.80) L30V/A60V/S64P/S86G Domain 2: ICOSL 201ICOS-CD28 N52H N57Y Q100P Domain 1: ICOSL WT 196 — 117853 (1.0) 70320(1.0) 7916 (1.0) 231 (1.0) Domain 2: Nkp30 WT 214 Domain 1: ICOSL 199ICOS-CD28 100396 (0.85) 83912 (1.19) 20778 (2.62) 228 (0.98) N52D Domain2: NKp30 215 B7-H6 L30V/A60V/S64P/S86G Domain 1: ICOSL 201 ICOS-CD2882792 (0.70) 68874 (0.98) 72269 (9.12) 561 (2.43) N52H/N57Y/Q100P Domain2: NKp30 215 B7-H6 L30V/A60V/S64P/S86G

TABLE 12 Stacked variant IgV Fc fusion proteins containing a CD80 IgVdomain and a ICOSL IgV domain Anti-CD3 Domain Structure SEQ CounterBinding Activity coimmobilization N terminal to C ID NO structure CD28MFI PD-L1 MFI ICOS MFI assay pg/mL IFN- terminal: domain (Ig selected(WT parental (WT parental (WT parental gamma (WT parental 1/domain 2/Fcdomain) against MFI ratio) MFI ratio) MFI ratio) IFN-gamma ratio) Domain1: CD80 WT 152 1230 (1.00) 2657 (1.00) 11122 (1.00) 69 (1.00) Domain 2:ICOSL WT 196 Domain 1: CD80 189 PD-L1 3383 (2.75) 4515 (1.70) 5158(0.46) 90 (1.30) E88D/K89R/D90K/A91G/ F92Y/K93R Domain 2: ICOSL 213ICOS/CD28 N52S/N57Y/H94D/L96F/ L98F/Q100R/G103E/F120S Domain 1: CD80 193PD-L1 2230 (1.81) 2148 (0.81) 3860 (0.35) 112 (1.62)A12T/H18L/M43V/F59L/ E77K/P109S/I118T Domain 2: ICOSL 213 ICOS/CD28N52S/N57Y/H94D/L96F/ L98F/Q100R/G103E/F120S Domain 1: CD80 193 PD-L15665 (4.61) 6446 (2.43) 15730 (1.41) 126 (1.83) A12T/H18L/M43V/F59L/E77K/P109S/I118T Domain 2: ICOSL 199 ICOS/CD28 N52D Domain 1: CD80 189PD-L1 6260 (5.09) 4543 (1.71) 11995 (1.08) 269 (3.90)E88D/K89R/D90K/A91G/ F92Y/K93R Domain 2: ICOSL 201 ICOS/CD28N52H/N57Y/Q100P Domain 1: CD80 193 PD-L1 3359 (2.73) 3874 (1.46) 8541(0.77) 97 (1.41) A12T/H18L/M43V/F59L/ E77K/P109S/I118T Domain 2: ICOSL201 ICOS/CD28 N52H/N57Y/Q100P Domain 1: ICOSL WT 196 3000 (1.00) 2966(1.00) 14366 (1.00) 101 (1.00) Domain 2: CD80 WT 152 Domain 1: ICOSL 213ICOS/CD28 3634 (1.21) 4893 (1.65) 6403 (0.45) 123 (1.22)N52S/N57Y/H94D/L96F/ L98F/Q100R/G103E/F120S Domain 2: CD80 189 PD-L1E88D/K89R/D90K/A91G/ F92Y/K93R Domain 1: ICOSL 213 ICOS/CD28 1095 (0.37)5929 (2.0) 7923 (0.55) 127 (1.26) N52S/N57Y/H94D/L96F/L98F/Q100R/G103E/F120S Domain 2: CD80 193 PD-L1 A12T/H18L/M43V/F59L/E77K/P109S/I118T Domain 1: ICOSL 199 ICOS/CD28 2023 (0.67) 5093 (1.72)16987 (1.18) 125 (1.24) N52D Domain 2: CD80 189 PD-L1E88D/K89R/D90K/A91G/ F92Y/K93R Domain 1: ICOSL 199 ICOS/CD28 3441 (1.15)3414 (1.15) 20889 (1.45) 165 (1.63) N52D Domain 2: CD80 193 PD-L1A12T/H18L/M43V/F59L/ E77K/P109S/I118T Domain 1: ICOSL 201 ICOS/CD28 7835(2.61) 6634 (2.24) 20779 (1.45) 95 (0.94) N52H/N57Y/Q100P Domain 2: CD80189 PD-L1 E88D/K89R/D90K/A91G/ F92Y/K93R Domain 1: ICOSL 201 ICOS/CD288472 (2.82) 3789 (1.28) 13974 (0.97) 106 (1.05) N52H/N57Y/Q100P Domain2: CD80 193 PD-L1 A12T/H18L/M43V/F59L/ E77K/P109S/I118T

TABLE 13 Stacked variant IgV Fc fusion proteins containing a CD80 orCD86 IgV domain and an ICOSL IgV domain SEQ ID Counter Binding ActivityFunctional Domain Structure NO structure PD-L1 MFI CTLA-4 MFI ActivityMLR N terminal to C terminal: (Ig selected (WT parental (WT parentalIFN-gamma domain 1/domain 2/Fc domain) against MFI ratio) MFI ratio)pg/mL Domain 1: CD80 WT 152 1230 11122 1756 Domain 2: ICOSL WT 196(1.00) (1.00) (1.00) Domain 1: CD86 WT 220196 29343 55193 6305 Domain 2:ICOSL WT (1.00) (1.00) (1.00) Domain 1: CD80 192 CD28 2280 3181 2281R29H/Y31H/T41G/Y87N/ (1.85) (0.29) (1.30) E88G/K89E/D90N/A91G/ P109SDomain 2: ICOSL 213 ICOS/CD28 N52S/N57Y/H94D/L96F/L98F/Q100R/G103E/F120S Domain 1: CD80 175 CD28 2309 26982 1561I67T/L70Q/A91G/T120S (1.88) (2.43) (0.89) Domain 2: ICOSL 213 ICOS/CD28N52S/N57Y/H94D/L96F/ L98F/Q100R/G103E/F120S Domain 1: CD80 192 CD28 428522744 1612 R29H/Y31H/T41G/Y87N/ (3.48) (2.04) (0.92)E88G/K89E/D90N/A91G/ P109S Domain 2: ICOSL 199 ICOS/CD28 N52D Domain 1:CD80 175 CD28 3024 16916 3857 I67T/L70Q/A91G/T120S (2.46) (1.52) (2.20)Domain 2: ICOSL 199 ICOS/CD28 N52D Domain 1: CD80 192 CD28 6503 72406886 R29H/Y31H/T41G/Y87N/ (5.29) (0.65) (3.92) E88G/K89E/D90N/A91G/P109S Domain 2: ICOSL 201 ICOS/CD28 N52H/N57Y/Q100P Domain 1: CD80 175CD28 3110 4848 3393 I67T/L70Q/A91G/T120S (2.53) (0.44) (1.93) Domain 2:ICOSL 201 ICOS/CD28 N52H/N57Y/Q100P Domain 1: CD86 221 CD28 11662 21165880 Q35H/H90L/Q102H (0.40) (0.38) (0.14) Domain 2: ICOSL 213 ICOS/CD28N52S/N57Y/H94D/L96F/ L98F/Q100R/G103E/F120S Domain 1: CD86 221 CD2824230 73287 1110 Q35H/H90L/Q102H (0.83) (1.33) (0.18) Domain 2: ICOSL199 ICOS/CD28 N52D Domain 1: CD86 221 CD28 1962 1630 587 Q35H/H90L/Q102H(0.07) (0.03) (0.09) Domain 2: ICOSL 201 ICOS/CD28 N52H/N57Y/Q100PDomain 1: ICOSL WT 196 3000 14366 4113 Domain 2: CD80 WT 152 (1.00)(1.00) (1.00) Domain 1: ICOSL WT 196 18005 53602 18393 Domain 2: CD86 WT220 (1.00) (1.00) (1.00) Domain 1: ICOSL 213 ICOS/CD28 10426 51286 18680N52S/N57Y/H94D/L96F/ (3.48) (3.57) (4.54) L98F/Q100R/G103E/F120S Domain2: CD80 192 CD28 R29H/Y31H/T41G/Y87N/ E88G/K89E/D90N/A91G/ P109S Domain1: ICOSL 213 ICOS/CD28 17751 29790 10637 N52S/N57Y/H94D/L9617 (5.92)(2.07) (2.59) L98F/Q100R/G103E/F120S Domain 2: CD80 175 CD28I67T/L70Q/A91G/T120S Domain 1: ICOSL 199 ICOS/CD28 2788 25870 6205 N52D(0.93) (1.80) (1.51) Domain 2: CD80 192 CD28 R29H/Y3IH/T41G/Y87N/E88G/K89E/D90N/A91G/ P109S Domain 1: ICOSL 199 ICOS/CD28 2522 13569 5447N52D (0.84) (0.94) (1.32) Domain 2: CD80 175 CD28 I67T/L70Q/A91G/T120SDomain 1: ICOSL 201 ICOS/CD28 9701 9187 5690 N52H/N57Y/Q100P (3.23)(0.64) (1.38) Domain 2: CD80 192 CD28 R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/ P109S Domain 1: ICOSL 213 ICOS/CD28 27050 212578131 N52S/N57Y/H94D/L96F/ (1.50) (0.40) (0.44) L98F/Q100R/G103E/F120SDomain 2: CD86 221 CD28 Q35H/H90L/Q102H Domain 1: ICOSL 199 ICOS/CD2834803 80210 6747 N52D (1.93) (1.50) (0.37) Domain 2: CD86 221 CD28Q35H/H90L/Q102H Domain 1: ICOSL 201 ICOS/CD28 5948 4268 26219N52H/N57Y/Q100P (0.33) (0.08) (1.43) Domain 2: CD86 221 CD28Q35H/H90L/Q102H

Example 9 Generation and Assessment of Engineered Cells Expressing aTransmembrane Immunomodulatory Protein

Engineered T cells were generated in which a transmembraneimmunomodulatory protein (TIP) containing an extracellular domain (ECD)containing either a variant CD80 as described above or an ICOSLaffinity-modified IgSF domain was co-expressed with a chimeric antigenreceptor (CAR). The TIP also contained a transmembrane domain and acytoplasmic domain of the corresponding wild-type CD80 or ICOSLtransmembrane protein sequence. The immunomodulatory activity of theengineered cells was compared to cells that only expressed the CAR orcells that co-expressed the corresponding wild-type CD80 or ICOSLtransmembrane protein with the CAR.

The exemplary CD80-TIP was a variant CD80 having an affinity-modifiedIgSF domain containing amino acid mutations in the IgV and IgC domainscorresponding to I67T/L70Q/A91G/T120S with reference to positions in theCD80 extracellular domain set forth in SEQ ID NO:28 and a transmembraneand cytoplasmic domain corresponding to residues 243-288 of SEQ ID NO:1.The amino acid sequence of the exemplary CD80-TIP is set forth in SEQ IDNO: 241 and is encoded by the sequence of nucleotides set forth in SEQID NO:242. The corresponding wild-type CD80 transmembrane protein hadthe sequence of amino acids set forth as amino acid residues 35-288 ofSEQ ID NO: 1 and encoded by the sequence of amino acids set forth in SEQID NO: 251.

The exemplary ICOSL-TIP was a variant ICOSL having an affinity-modifiedIgSF domain containing amino acid mutations in the IgV domaincorresponding to N52H/I143T with reference to positions in the ICOSLextracellular domain set forth in SEQ ID NO:32 and a transmembrane andcytoplasmic domain corresponding to residues 257-302 of SEQ ID NO:5. Theamino acid sequence of the exemplary ICOSL-TIP is set forth in SEQ IDNO:243 and is encoded by the sequence of nucleotides set forth in SEQ IDNO:244. The corresponding wild-type ICOSL transmembrane protein had thesequence of amino acids set forth as amino acid residues 19-302 of SEQID NO:5 and encoded by the sequence of amino acids set forth in SEQ IDNO: 252.

The TIP containing the affinity-modified domain or the wild-typetransmembrane protein containing a corresponding non-affinity modifiedIgSF domain were co-expressed in T cells with a 1^(st) generationchimeric antigen receptor (CAR) containing a CD3zeta intracellularsignaling domain. The 1^(st) generation CAR included an scFv specificfor CD19 (SEQ ID NO:245), a hinge and transmembrane domain derived fromCD8 (SEQ ID NO:246) and an intracellular signaling domain derived fromCD3zeta (set forth in SEQ ID NO:47). The nucleotide sequence encodingthe CD19 scFv-CD3zeta CAR is set forth in SEQ ID NO: 248 and the aminoacid sequence of the CD19 scFv-CD3zeta CAR is set forth in SEQ ID NO:479.

Nucleic acid molecules encoding the CAR alone or also encoding one ofthe exemplary TIPs or wild-type transmembrane proteins separated fromthe CAR by a self-cleaving T2A sequence (SEQ ID NO:250 and encoded bythe sequence of nucleotides set forth in SEQ ID NO:249) were generated.Exemplary constructs contained nucleic acid sequences set forth in Table14. As a control, a nucleic acid construct encoding a 2^(nd) generationCAR additionally containing a CD28 costimulatory domain also wasgenerated (CD19 scFv-CD28-CD3zeta).

TABLE 14 Nucleic Acid Constructs CAR T2A Linker TIP (SEQ ID NO) (SEQ IDNO) (SEQ ID NO) CD19 scFv - + − − CD3zeta (248) CD19 scFv - + + WildtypeCD80 CD3zeta - T2A - (248) (249) (251) B7-1 CD19 scFv - + + CD80 TIPCD3zeta - T2A - (248) (249) (242) B7-1_TIP CD19 scFv - + + WildtypeICOSL CD3zeta - T2A - (248) (249) (252) ICOSL CD19 scFv - + + ICOSL TIPCD3zeta - T2A - (248) (249) (244) ICOSL_TIP

The nucleic acid molecules were individually cloned into a lentiviralvector, which was used to transduce T cells isolated from human PBMCsamples obtained from three different healthy donors. Lentivirusparticles containing the nucleic acid sequences were produced afterco-transfection of HEK293 cells with the vectors and lentiviruspackaging constructs. The lentivirus particles were collected from theculture medium by ultracentrifugation and titered by qRT-PCR. Humanperipheral blood mononuclear cells (PBMC) were isolated from threenormal blood donors using density sedimentation. The PBMC were culturedovernight with anti-CD3 and anti-CD28 antibodies and IL-2, thentransduced with the lentivirus preparations at a multiplicity ofinfection of 5:1. The lentiviral vectors encoding the control 2^(nd)generation CAR was only used to transduce cells from one donor.

After two weeks (14 days) of culture, the cells were analyzed forcytotoxicity following co-culture with target antigen-expressing cellsusing the Acea Real-Time Cell Analyzer (RTCA), which measures theimpedance variations in the culture media of a 96-well microelectronicplate (E-plate), and shows the changes in cell number and morphology ina real-time plot. CD19-expressing HeLa target cells (HeLa-CD19) wereseeded into a 96-well E-plate and the impedance of each monolayer wasmonitored for 24 hours using the RTCA system. The engineered T cellswere added to the wells at an effector:target ratio of 10:1 and thewells were monitored for another 48 hours. The results were displayedand recorded as Cell Index (CI) value derived from the change inmeasured electrical impedance and were then ratio transformed bydividing the CI readouts of all wells at all time points over the CIvalue of individual wells at a same time (base-time) to obtain anormalized cell index value representing the percentage of the value atthe base-time (see Zhang et al. “Introduction to the Data Analysis ofthe Roche xCELLigence® System with RTCA Package.” Bioconductor. May 3,2016,bioconductor.org/packages/devel/bioc/vignettes/RTCA/inst/doc/aboutRTCA.pdf.Accessed Sep. 9, 2016). In this assay, a decrease in the impedance of amonolayer reflects killing of the target cells by the transduced cells.

The results showed that decreased impedance was observed in cellsexpressing the 1^(st) generation CAR compared to non-transduced T cells,although the degree of decreased impedance for cells expressing the1^(st) generation CAR was less than cells expressing the 2^(nd)generation CAR. The decreased impedance in cells expressing the 1^(st)generation CAR continued generally for up to the first 8 hours of theassay, while only the 2nd generation CAR-expressing cells continued todecrease the impedance thereafter.

As shown in FIG. 1, in one donor, each of the cells co-expressing theTIP or corresponding wild-type transmembrane protein with the 1^(st)generation CAR exhibited a greater decrease in impedance, indicatinggreater cytotoxic activity, compared to cells only expressing the 1^(st)generation CAR. Further, the results showed that the cytotoxic activitywas greater in CAR-expressing cells that co-expressed the CD80-TIP orICOSL-TIP relative to CAR-expressing cells that co-expressed thecorresponding wild-type CD80 or ICOSL transmembrane proteins containinga non-affinity modified IgSF domain. The observed results of theseTIP-engineered cells showed that cytotoxic activity in cellsco-expressing the CD80-TIP or ICOSL-TIP with the CAR exhibit increasedactivity to modulate the cytotoxic immune response of antigen-specific Tcells, such as the CAR-expressing T cells.

In the other two donors, the cells expressing the CD80-TIP did notresult in a greater decreased impedance compared to cells expressing thecorresponding wild-type CD80 transmembrane protein. In one donor, therewere not enough cells to transduce with the wild-type transmembraneprotein construct, although in this donor the ICOS-L TIP gave the bestcytotoxicity compared to the other constructs tested. In the otherdonor, the cells expressing the ICOS-L-TIP did not result in a greaterdecreased impedance compared to cells expressing the correspondingwild-type ICOS-L transmembrane protein. In the tested cells, all cellsco-expressing either a CD80-TIP, ICOSL-TIP or corresponding wild typetransmembrane protein with the CAR exhibited greater cytotoxic activitythan cells only expressing the 1st generation CAR. The differences inthe results observed among donors may be related to the differences inthe T cells among the donors, differences in expression levels of thevarious engineered proteins on the surface of the cells, the particularconditions used in this exemplary assay for assessing killing in cells(e.g. assessing Day 14 transduced cells, assessing a singleeffector:target cell ratio) or other factors.

Example 10 Assessment of Binding and Activity of ICOSL IgSF DomainVariants

Additional ECD ICOSL variants were identified by the yeast selectionmethod substantially as described above and were used to produce ECD-Fcfusion proteins as described in Example 5. Binding studies wereperformed to assess specificity and affinity of ICOSL domain variantimmunomodulatory proteins for cognate binding partners substantially asdescribed in Example 6.

A. Binding and Functional Characterization

Binding was assessed to cells expressed full-length cognate bindingpartners CD28, ICOS and CTLA-4 substantially as described in Example 6.Bioactivity of the ECD ICOSL variants also was assessed in an anti-CD3coimmobilization assay or human Mixed Lymphocyte Reaction (MLR)substantially as described in Example 6, except that for thecoimmobilization assay, costimulatory activity was determined by cultureof human T cells with a mixture of 10 nM plate-bound anti-CD3 and 40 nMICOSL Fc variant proteins.

Table 15 depicts exemplary results for the additional ICOSL IgSF domainvariants for binding to cell-expressed counter structures andbioactivity in the anti-CD3 coimmobilization assay or MLR assay. Theexemplary amino acid substitutions depicted in Table 15 are designatedby amino acid position number corresponding to the respective reference(e.g., unmodified) ICOSL ECD sequence set forth in SEQ ID NO:32. Theamino acid position is indicated in the middle, with the correspondingunmodified (e.g. wild-type) amino acid listed before the number and theidentified variant amino acid substitution listed after the number.Column 2 sets forth the SEQ ID NO identifier for the variant ECD foreach variant ECD-Fc fusion molecule.

The results in Table 15 depict binding activity as measured by the MeanFluorescence Intensity (MFI) value for binding of each variant Fc-fusionmolecule to cells engineered to express the cognate counter structureligand and the ratio of the MFI compared to the binding of thecorresponding reference (e.g., unmodified) ECD-Fc fusion molecule notcontaining the amino acid substitution(s) to the same cell-expressedcounter structure ligand. The functional activity of the variantFc-fusion molecules to modulate the activity of T cells also is shownbased on the calculated levels of IFN-gamma in culture supernatants(pg/mL) generated either i) with the indicated variant ECD-Fc fusionmolecule coimmoblized with anti-CD3 or ii) with the indicated variantECD-Fc fusion molecule in an MLR assay. The Table also depicts the ratioof IFN-gamma produced by each variant ECD-Fc compared to thecorresponding unmodified (parental) ECD-Fc in both functional assays.

The results show altered, including increased, binding affinity ofaffinity-modified ICOSL IgSF domain variants for at least one cognatecounter structure ligand and/or improved immunological activity.Specifically, similar to the initial hits identified in Example 6, theselections resulted in the identification of a number of additionalICOSL IgSF domain variants that were affinity-modified to exhibitincreased binding for at least one, and in some cases more than one,cognate counter structure ligand. In addition, the results showed thataffinity modification of the variant molecules also exhibited improvedactivities to both increase and/or decrease immunological activitydepending on the format of the molecule as described in Example 6.

TABLE 15 ICOSL variants: binding data and costimulatory bioactivitydata. Anti-CD3 IFN-gamma MLR ICOS CD28 CTLA-4 Coimmobilization IFN-gammaSEQ tfxn MFI tfxn MFI tfxn MFI Assay pg/mL pg/mL ID NO (parental(parental (parental (parental (parental ICOSL mutation(s) (ECD) ratio)ratio) ratio) ratio) ratio) N52H, F78L, Q100R, C198R 373 9568 (0.12)1966 (0.24) 1454 (0.12) 130 (0.31) 5927 (1.84) N52H, N57Y, Q100R, 3649418 (1.16) 136665 (16.55) 115352 (9.59) 944 (2.21) 821 (0.25) V110D,C198R, S212G N52H, N57Y, R75Q, Q100P, 374 5558 (0.07) 7465 (0.90) 4689(0.39) 122 (0.28) 1136 (0.35) V110D N52H, N57Y, Q100R, C198R 365 9148(1.13) 134923 (16.33) 83241 (6.92) 1060 (2.48) 375 (0.12) N52H, N57Y,L74Q, V110D, 375 9448 (1.17) 128342 (15.54) 123510 (10.26) 1137 (2.66)889 (0.28) S192G N52H, Q100R 285 9478 (1.17) 151977 (18.40) 133929(11.13) 972 (2.28) 794 (0.25) N52H, S121G, C198R 376 9128 (1.13) 124732(15.10) 182607 (15.18) 827 (1.94) 1257 (0.39) A20V, N52H, N57Y, Q100R,287 5828 (0.72) 76973 (9.32) 73640 (6.12) 447 (1.05) 2283 (0.71) S109GN52H, N57Y, Q100P, C198R 461 9548 (1.18) 130676 (15.82) 81966 (6.81)1125 (2.64) 643 (0.20) N52H, N57Y, R61S, Q100R, 289 1018 (0.13) 9129(1.11) 5790 (0.48) 109 (0.25) 5094 (1.58) V110D, L173S N52H, N57Y,Q100R, 290 9978 (1.23) 137372 (16.63) 70764 (5.88) 1316 (3.08) 473(0.15) V122A N52H, N57Y, Q100R, F172S 291 1028 (1.27) 135821 (16.44)73320 (6.09) 1561 (3.66) 486 (0.15) N52H, N57Y, Q100R 283 9858 (1.22)140612 (17.02) 75106 (6.24) 1648 (3.86) 778 (0.24) N52S, F120S, N227K377 9438 (1.17) 67796 (8.21) 82370 (6.85) 1157 (2.71) 1626 (0.50) N52S,N194D 366 9798 (1.21) 59431 (7.19) 74502 (6.19) 1671 (3.91) 1690 (0.52)N52S, V97A 294 3138 (0.04) 1733 (0.21) 1541 (0.13) 84 (0.20) 3858 (1.20)N52S, F120S 293 9068 (1.12) 67233 (8.14) 97880 (8.13) 1178 (2.76) 2814(0.87) N52S, G72R 295 9288 (1.15) 51638 (6.25) 62339 (5.18) 1161 (2.72)2947 (0.91) N52S, A71T, A117T, T190A, 378 8918 (1.10) 44044 (5.33) 56646(4.71) 1076 (2.52) 4031 (1.25) C198R N52S, E220G 297 3878 (0.05) 2047(0.25) 1796 (0.15) 122 (0.29) 1927 (0.60) Y47H, N52S, V107A, F120S 2983268 (0.04) 2562 (0.31) 2104 (0.17) 334 (0.78) 4390 (1.36) WT ICOSL 328088 (1.00) 8260 (1.00) 12033 (1.00) 427 (1.00) 3226 (1.00) T43A, N52H,N57Y, L74Q, 379 2821 (0.02) 2180 (0.49) 2051 (0.12) 184 (0.75) D89G,V110D, F172S N52H, N57Y, Q100R, V1071, 381 174586 (0.97) 122383 (27.24)76202 (4.31) 985 (4.01) 1037 (0.36) V110D, S132F, I154F, C198R, R221GE16V, N52H, N57Y, Q100R, 300 190765 (1.05) 129070 (28.73) 68488 (3.87)4288 (17.46) 1225 (0.43) V110D, H115R, Y152C, K156M, C198R Q37R, N52H,N57Y, Q100R, 301 148638 (0.82) 91104 (20.28) 13498 (0.76) 62 (0.25) 7643(2.68) V110N, S142F, C198R, D217V, R221G N52H, N57Y, Q100R, 302 179194(0.99) 123312 (27.45) 84136 (4.76) 762 (3.10) 1342 (0.47) V110D, C198RN52H, N57Y, Q100R, 303 5236 (0.03) 4160 (0.93) 3305 (0.19) 49 (0.20)2039 (0.72) V110D, V116A, L161M, F172S, S192G, C198R F27S, N52H, N57Y,V110N 304 20154 (0.11) 8613 (1.92) 3903 (0.22) 83 (0.34) 7522 (2.64)F27S, N52H, N57Y, V110N 304 5236 (0.03) 4160 (0.93) 2957 (0.17) 40(0.16) — N52S, H94E, L96I, S109N, 305 198604 (1.10) 100361 (22.34)102892 (5.82) 1253 (5.10) 5645 (1.98) L166Q, S18R, N52S, F93L, I143V,306 154561 (0.85) 7625 (1.70) 4254 (0.24) 203 (0.83) 5239 (1.84) R221GA20T, N52D, Y146C, Q164L 307 149661 (0.83) 9073 (2.02) 6901 (0.39) 287(1.17) 4829 (1.69) V11E, N30D, N52H, N57Y, 308 180016 (1.00) 120230(26.76) 62809 (3.55) 2218 (9.03) 7283 (2.56) H94E, L96I, L98F, N194D,V210A, I218T N52S, H94E, L96I, V122M 309 198717 (1.10) 88901 (19.79)94231 (5.33) 590 (2.40) 618 (0.22) N52H, N57Y, H94E, L96I, 310 87711(0.48) 42035 (9.36) 31798 (1.80) 67 (0.27) 2500 (0.88) F120I, S126T,W153R, I218N M10V, S18R, N30D, N52S, 311 180665 (1.00) 64929 (14.45)48362 (2.73) 1193 (4.86) 13647 (4.79) S126R, T139S, L203F S25G, N30D,N52S, F120S, 312 178834 (0.99) 66127 (14.72) 46631 (2.64) 1246 (5.07)2202 (0.77) N227K N30D, N52S, L67P, Q100K, 313 18630 (0.10) 1986 (0.44)1940 (0.11) 54 (0.22) 2752 (0.97) D217G, R221K, T225S WT ICOSL 32 180900(1.00) 4493 (1.00) 17685 (1.00) 246 (1.00) 2850 (1.00) R26S, N52H, N57Y,V110D, 908 N/A N/A N/A N/A N/A T137A, C198R N52H, N57Y, Q100R, 314 2831(0.04) 2881 (0.57) 2464 (0.23) 59 (0.08) — V110D, A117T, T190S, C198RN52H, N57Y, Q100R, 315 58478 (0.79) 74031 (14.75) 56850 (5.33) 712(0.96) 1093 (0.23) V110D, F172S, C198R S25G, F27C, N52H, N57Y, 316 22514(0.30) 21320 (4.25) 20450 (1.92) 353 (0.48) 5765 (1.21) Q100R, V110D,E135K, L173S, C198R N52H, N57Y, V110A, 317 84236 (1.14) 81842 (16.31)121519 (11.39) 4593 (6.18) 1137 (0.24) C198R, R221I M10I, S13G, N52H,N57Υ, 318 6362 (0.09) 6001 (1.20) 4834 (0.45) 141 (0.19) 4326 (0.91)D77G, V110A, H129P, I143V, F172S, V193M, C198R N52H, N57Υ, R61C, Y62F,319 4355 (0.06) 4316 (0.86) 3430 (0.32) 110 (0.15) 6854 (1.44) Q100R,V110N, F120S, C198R N52H, N57Y, Q100R, 367 96736 (1.31) 77881 (15.52)148012 (13.88) 8765 (11.79) 630 (0.13) L102R, V110D, H115R, C198R N52H,N57Y, Q100R, 321 67578 (0.91) 64953 (12.94) 95731 (8.98) 1672 (2.52)1490 (0.31) V110D, N144D, F172S, C198R N52S, H94E, L98F, Q100R, 32280690 (1.09) 78750 (15.69) 148160 (13.89) 3564 (4.80) 1497 (0.32) N52S,E90A 323 108908 (1.47) 31086 (6.19) 108866 (10.21) 4564 (6.14) 3927(0.83) N30D, K42E, N52S 324 85726 (1.16) 4293 (0.86) 10755 (1.01) 5211(7.01) 5656 (1.19) N52S, F120S, I143V, I224V 325 90862 (1.23) 28443(5.67) 105229 (9.87) 4803 (6.46) 4357 (0.92) WT ICOSL 32 73964 (1.00)5018 (1.00) 10665 (1.00) 743 (1.00) 4748 (1.00)

B. Cytokine Production in Anti-CD3 Costimulation Assays

Exemplary variant ECD ICOSL Fc-fusion molecules described above werefurther assessed for stimulation of cytokines IL-17 in the anti-CD3costimulatory (coimmobilization) bioactivity assay described above. Amixture of 10 nM plate-bound anti-CD3 and 40 nM ICOSL Fc variantproteins were cultured with human T cells. Supernatants were collectedand IL-17 levels were determined by ELISA. The amount of IL-17 inculture supernatants (pg/mL) generated with the indicated variant ECD-Fcfusion molecule and corresponding unmodified (parental) ECD-Fccoimmobilized with anti-CD3 was measured. For comparison, also shown inthis Table are the results for production of IFN-gamma in the same assayas depicted in Table 15 for the exemplary variants.

Results are shown in Table 16, which depict the pg/mL of IL-17 measuredin the supernatant as well as the ratio (fold increase) of IL-17produced by each variant ECD-Fc compared to the corresponding unmodified(wild-type) ECD-Fc. Similar results are shown for IFN-gamma. Also shownis the % of total IL-17 or IFN-gamma cytokine produced by cells. Theresults showed that affinity modification of the variant moleculesexhibited altered functional T cell activity to increase IL-17 inaddition to IFN-gamma in the costimulation assay.

TABLE 16 Costimulatory Bioreactivity Data for ICOSL IgSF Domain Variants% of Total Cytokine % SEQ IL-17A IFN-g Total Produced Total ICOSL ID NOIL-17A Fold IFN-g Fold Fold % % IL-17 + mutation(s) (ECD) [pg/mL] ↑WT[pg/mL] ↑WT ↑WT IL-17 IFN-g IFN-g N52H, N57Y, 365 617 7.93 1060 2.4810.42 5.51 0.77 6.28 Q100R, C198R N52H, N57Y, 290 647 8.33 1316 3.0811.41 5.79 0.96 6.75 Q100R, V122A N52H, N57Y, 291 549 7.06 1561 3.6610.72 4.91 1.14 6.05 Q100R, F172S N52Y, N57Y, 112 90 1.05 1999 2.69 3.740.81 2.91 3.72 F138L, L203P V11E, N30D, N52H, 308 319 3.16 2218 9.0312.19 2.85 3.23 6.08 N57Y, H94E, L96I, L98F, N194D, V210A, I218T N52H,N57Y, 367 510 5.90 8765 11.79 17.70 4.56 12.78 17.33 Q100R, L102R,V110D, H115R, C198R N52H, N57Y, Q100R 283 473 6.08 1648 3.86 9.94 4.231.20 5.43 N52H, Q100R 285 358 4.60 972 7.01 11.62 3.20 0.71 3.91 N52H,N57Y, 364 124 1.60 944 2.21 3.81 1.11 0.69 1.80 Q100R, V110D, C198R,S212G N52H, N57Y, Q100P 113 127 1.47 4922 6.62 8.09 1.14 7.17 8.31 E16V,N52H, N57Y, 300 22 7.11 130 17.46 24.57 6.41 6.25 12.66 Q100R, V110D,H115R, Y152C, K156M, C198R N30D, K42E, N52S 324 349 4.04 5211 7.01 11.053.12 7.60 10.71 N52S, F120S, I143V, 325 292 3.39 4803 6.46 9.85 2.617.00 9.62 I224V N52S, E90A 323 306 3.54 4564 6.14 9.68 2.73 6.65 9.39N52H, N57Y, 317 290 3.35 4593 6.18 9.53 2.59 6.69 9.28 V110A, C198R,R221I N52S, N194D 366 428 5.50 1671 3.90 9.4 1.52 5.19 5.40 N52H, I143T135 84 — 1727 — 3.30 0.75 2.52 3.27 N52D 111 126 — 1447 — 3.41 1.13 2.113.23

Example 11 Generation of Additional Engineered T Cell Expressing aTransmembrane Immunomodulatory Protein and Assessment of Proliferation

This Example describes the generation of additional engineered T cellsin which a transmembrane immunomodulatory protein (TIP) containing anextracellular domain (ECD) containing ICOSL affinity-modified IgSFdomain was co-expressed with a chimeric antigen receptor (CAR).Specifically, the TIP was generated to include the ECD of exemplaryvariant ICOSL containing amino acid mutations N52D, N52H/N57Y/Q100P,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R or N52H/N57Y/Q100Rwith reference to positions in the ICOSL extracellular domain set forthin SEQ ID NO:32. The TIP also contained a transmembrane domain and acytoplasmic domain of the corresponding wild-type ICOSL transmembraneprotein sequence corresponding to residues 257-302 of SEQ ID NO:5. Thesequence of the TIP with and without its signal peptide are as follows:N52D (SEQ ID NO: 496 and 497); N52H/N57Y/Q100P (SEQ ID NO: 498 and 499);E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R (SEQ ID NO: 500 and501) and N52H/N57Y/Q100R (SEQ ID NO: 502 and 503). For comparison, thefull-length transmembrane wild-type ICOSL (amino acid residues 19-302 ofSEQ ID NO:5) also was expressed in cells. The sequence of the wildtypeTIP with and without its signal peptide is set forth in SEQ ID NO: 494and 495. The nucleic acid encoding the TIP also included a sequenceencoding a green fluorescent protein (GFP) separated from the TIP by aself-cleaving T2A sequence.

The TIP containing the affinity-modified domain or the wild-typetransmembrane protein containing a corresponding non-affinity modifiedIgSF domain were co-expressed in T cells with a chimeric antigenreceptor (CAR). The nucleotide sequence encoding the CAR encodes, inorder: a CD8 signal sequence (SEQ ID NO: 481), an anti-CD19 scFv (SEQ IDNO: 482), a hinge/transmembrane region derived from CD8 (SEQ ID NO:483), a costimulatory signaling domain derived from 4-1BB (SEQ ID NO:484), and a CD3zeta signaling domain (SEQ ID NO: 247). The resultinganti-CD19 CAR has the sequence of amino acids set forth in SEQ ID NO:490. The nucleic acid encoding the CAR also included a sequence encodinga blue fluorescent protein (BFP; SEQ ID NO: 489) separated from the CARby a self-cleaving T2A sequence (set forth in SEQ ID NO: 488).

Viral vector constructs were separately generated into which was clonedeither the nucleic acid molecule encoding the CAR alone or a nucleicacid molecule encoding one of the exemplary TIPs or wild-type ICOSL. Theviral vector encoding the CAR and the viral vector encoding the TIP orwild-type ICOSL were co-transduced into T cells. For transduction,primary T cells were activated with anti-CD3 and anti-CD28 beads (Dynal)at 1:1 bead:cell ratio and incubated in the presence of 100 IU/mL ofIL-2 at 37° C. for 2 days. T cells were then harvested and transducedwith 400 μL of CAR viral supernatant and 400 μL of TIP viral supernatantin the presence of 8 g/mL of polybrene. The cells were spinoculated at1000 g for 30 minutes at 30° C. The cells were then transferred andincubated overnight at 37° C. After the incubation, cells were collectedand viral supernatant was removed. The cells were resuspended withcomplete media and 50 IU/mL of IL-2. Cells were expanded, replenishedwith IL-2 and media every two days for a total of 6 days. Beads wereremoved from the cells using a magnet and counted before being assessedin a proliferation assay. An exemplary expression profile of a TIP andCAR in an exemplary transduced T cells is shown in FIG. 2A.

To assess proliferation of CAR T cells and CAR-TIP T cells in responseto antigen, cells were labeled with cell trace far red dye.CD19-expressing Nalm6 target cells were titrated starting from 1.5:1target:T cell ratio and by 1:2 dilutions with 8-point dilution. LabeledCAR T cells or CAR-TIP T cells were added to Nalm6 cells and the culturewas incubated for 4 days before cells were analyzed by flow cytometry.The supernatant was collected and further assessed in a cytokine releaseassay.

As shown in FIG. 2B, CAR+ primary T cells proliferate in a dosedependent manner to CD19+ NALM6 cells. Compared to a CAR only T cells, Tcells coexpressing the CAR and either wild-type ICOSL or one of theexemplary ICOSL TIP exhibited enhanced proliferation compared to CARonly expressing T cells. Co-expression of a CAR and a TIP containingeither the N52H/N57Y/Q100P,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R or N52H/N57Y/Q100Rvariant ICOSLECD exhibited greater proliferation than T cellsco-expressing the CAR and wildtype ICOSL, indicating that TIPs expressedon primary T cells provide an improved costimulatory signal to enhance Tcell proliferation.

Example 12 Purification and Assessment of Purified of ICOSL IgSF DomainVariants

A purification strategy was employed for exemplary candidate hitsdescribed in Example 6 and 10. Human cells derived from the 293 cellline (Expi293) were transiently transfected with expression constructand the ECD ICOSL Fc fusion molecule was expressed in the cells. The Fcfusion proteins were then purified from supernatants with Protein A byaffinity chromatograpy (MabSelect SuRe). This initial purification stepwas then followed by a preparative size exclusion chromatography (SEC)step to further purify the proteins (Superdex200 16×60). Samples fromboth purification steps were retained and compared by analytic SEC. Theconcentration of the protein was determined after Protein Apurification. The resulting purified proteins also were analyzed byanalytic SEC on a high performance liquid chromatography (HPLC) toassess purity.

The percent main peak in the purified samples was determined andcompared to protein purified in the initial Protein A step (% Main PeakProt A pool) versus protein purified with Protein A followed bypreparative SEC (% Main Peak SEC pool T=D0). As shown in Table 17, theadditional SEC step substantially increased protein purity of purifiedproteins. To further assess stability of the proteins, proteins purifiedby preparative SEC were left at room temperature for 24 hours and then %Main Peak by HPLC (% Main Peak SEC pool T=D24) was assessed and comparedto D0 sample. The change in % Main Peak at D0 versus D24 was determined(▴% Main Peak SEC pool). As shown in Table 17, most of the testedexemplary variant ECD ICOSL Fc fusion molecules exhibited little changein % Main Peak at this time, indicating minimal aggregation of theprotein variants had occurred.

TABLE 17 Purification of ICOSL Protein Variants Expi293 % Main % Main %Main SEQ ID Prod. Peak Peak SEC Peak SEC ▴ % Main NO Prot A Prot A poolpool Peak SEC ICOSL mutation(s) (ECD) mg/L pool T = D 0 T = D 24 poolN52S, N194D 366 120 87.9 93.5 92 1.5 N52H, N57Y, Q100R, F172S 291 21786.9 97.4 95.6 1.8 N52S, E90A 323 128 86.5 89.5 88.3 1.2 N52H, Q100R 285176 85.9 97.5 96.1 1.4 N52H, N57Y, Q100R 283 186 85.1 97.6 95.7 1.9N52S, F120S, I143V, I224V 325 87 83.2 88.9 88.3 0.6 N52H, N57Y, Q100R,C198R 365 204 82.9 95.8 92.3 3.5 N52H, N57Y, Q100P 113 63 80.5 94.5 88.56 N30D, K42E, N52S 324 81 80 95.4 91.3 4.1 N52H, N57Y, Q100R, L102R, 367141 78.9 96 92.9 3.1 V110D, H115R, C198R N52H, N57Y, Q100R, V122A 290260 77.6 96.4 95.2 1.2 N52Y/N57Y/F138L/L203P 112 40 75.6 96.8 94.8 2E16V, N52H, N57Y, Q100R, 300 60 73.8 97.1 95.8 1.3 V110D, H115R, Y152C,K156M, C198R N52H, N57Y, V110A, C198R, 317 95 65.4 90.9 86 4.9 R221IN52H, N57Y, Q100R, V110D, 364 73 50.6 87.9 78.6 9.3 C198R, S212G V11E,N30D, N52H, N57Y, H94E, 308 58 — — — — L96I, L98F, N194D, V210A, I218TN52H, I143T 135 134 93.2 96 92.7 3.3 N52D 111 136 90.4 95.5 93.3 2.2

Example 13 Assessment of Costimulatory Bioactivity of Purified ICOSLIgSF Domain Variant Hits

Exemplary ECD ICOSL Fc fusion molecules purified as described in Example12 were assessed for bioactivity by MLR substantially as described inExample 6. A mixture of 10 nM or 40 nM ICOSL Fc variant proteins wasbound overnight to 96-well plates in the presence of 10 nM anti-CD3. Theplates were washed and 100,000 CFSE labelled pan T cells were added for96 hours. Supernatants were collected, and IFN-gamma and IL-17 levelswere measured by ELISA.

Results for the cytokine secretion induced by anti-CD3 costimulationwith exemplary tested variants (10 nM and 40 nM ICOSL Fc) are shown inFIGS. 3A and 3B, which indicates exemplary IgSF domain amino acidsubstitutions (replacements) in the ECD of ICOSL. The bar graphs inFIGS. 3A and 3B depict the amount of secreted IFN-gamma and IL-17,respectively, by ELISA in the supernatants (pg/mL). The level ofcytokine release induced by anti-CD3 costimulation with the testedvariants compared to the level induced by anti-CD3 costimulation with WTICOSL is indicated by the horizontal line. The results showed thataffinity modification of the variant molecules exhibited activity tomodulate functional T cell activity, including to substantially increaseIFN-gamma and IL-17 secretion in the costimulation assay. Increasedimmunological activity was observed with some variants.

Example 14 Assessment of Proliferation of Purified ICOSL IgSF DomainVariant Hits

Exemplary variant ECD ICOSL Fc fusion molecules purified as described inExample 12 were assessed for ability to costimulate anti-CD3-inducedproliferation of T cells. Primary T cells were labeled withcarboxyfluorescein succinmidyl ester (CFSE). A mixture of 10 nM or 40 nMvariant ECD ICOSL Fc or wild-type ECD ICOSL proteins were boundovernight to 96-well plates in the presence of 10 nM anti-CD3, and thenlabeled T cells were added and incubated for 3 days. As a control,proliferation also was assessed in the presence of bound anti-CD3 andIgG or IgG alone. Cells were stained for CD4 or CD8 surface markers andproliferation of total T cells, CD4+ T cells or CD8+ T cells wasdetermined by assessing CFSE dilution by flow cytometry.

The results are set forth in FIG. 4A and FIG. 4B for exemplary variantstested at 40 nM and 10 nM ICOSL, respectively. As shown in FIG. 4A,nearly all tested variant ECD ICOSL Fc fusion molecules inducedproliferation greater than WT control. As shown in FIG. 4B, differencesin proliferation were more apparent at 10 nM with certain variantsproviding maximal proliferation even at this lower concentration.

Example 15 Assessment of Binding and Activity of Purified ICOSL IgSFDomain Variant Hits

Exemplary variant ECD ICOSL Fc fusion molecules purified as described inExample 12 were assessed for binding and functional activities usingmethods substantially as described in Example 6 or Example 10.

A. Flow Cytometric Binding Assays

Human cells derived from the 293 cell line (Expi293) were transfectedwith CD28, CTLA-4, ICOS or mock transfected. Cells were then incubatedwith ECD ICOSL Fc fusion molecules or wild-type ECD ICOSL-Fc that weretitrated from 100,000 pM to 46 pM, and binding was observed using aPE-conjugated anti-human Fc as described in Example 6. Binding wasassessed by flow cytometry and mean fluorescence intensity (MFI) andpercent (%) of cells positive for signal was determined using Cell QuestPro software (Becton Dickinson, USA). The concentration of ICOSL-Fc thatgave a half-maximal MFI response (MFI EC50) or % positive cells (% (+)EC50) was determined.

Table 18 sets forth the results. The ICOSL amino acid substitutionsdepicted in Table 18 are designated by amino acid position numbercorresponding to the respective reference (e.g., unmodified) ICOSL ECDsequence set forth in SEQ ID NO:32. For some values (e.g. WT binding toCD28) it was not possible to obtain an EC50, therefore 1000000 pM wasarbitrarily piced for data formatting purposes. Similar to resultsobtained from previous binding assays as described in Example 10 above,altered binding affinity of variant ICOSL ECD-Fc fusion molecule for atleast one cognate counter structure ligand was observed.

TABLE 18 Flow Cytometric EC50s for ICOSL variants CD28 CD28 CTLA-4CTLA-4 ICOS ICOS SEQ MFI % (+) MFI % (+) MFI % (+) ID NO EC50 EC50 EC50EC50 EC50 EC50 ICOSL mutation(s) (ECD) [pM] [pM] [pM] [pM] [pM] [pM] WTICOSL 32 1000000 1000000 1000000 1000000 10543 762 N52H, I143T 135 19147567 20259 1891 2666 286 N52H, N57Y, Q100R, C198R 365 950 159 73548 4221032 179 N52H, N57Y, Q100R, V122A 290 29701 152 1008 293 302 64 N52H,N57Y, Q100R, F172S 291 1006 231 1332 396 779 130 N52Y/N57Y/F138L/L203P112 7844 386 7457 994 3104 408 V11E, N30D, N52H, N57Y, H94E, 308 5961595 6909 1026 5514 852 L96I, L98F, N194D, V210A, I218T N52H, N57Y,Q100R, L102R, 367 1034 307 23328 579 3172 347 V110D, H115R, C198R N52H,N57Y, Q100R 283 1665 238 11002 533 383 131 N52H, Q100R 285 1305 274 85931997 702 167 N52H, N57Y, Q100R, V110D, 364 4987 594 30382 922 50219 814C198R, S212G N52H, N57Y, Q100P 113 21137 402 22651 758 4090 320 E16V,N52H, N57Y, Q100R, 300 2508 387 5399 806 2381 421 V110D, H115R, Y152C,K156M, C198R N30D, K42E, N52S 324 — 3683800 8593 1997 3251 558 N52S,F120S, I143V, I224V 325 902400 9060 28126 2948 4366 245 N52S, E90A 3231339700 31302 31419 5828 5225 473 N52H, N57Y, V110A, C198R, 317 1809 4267201 841 1293 433 R221I N52S, N194D 366 944669 11876 1254880 5170 473206 N52D 111 288617 17793 396841 3891 2642 137

B. ForteBio Binding Assay

Protein-protein interactions between the receptors and ICOSL domainvariant immunomodulatory proteins were further assessed using Fortebiobinding assays. ICOS, CD28, and CTLA-4 receptors were loadedindividually onto anti-human capture sensors (ForteBio Octet AHC) andwildtype unmodified ICOSL ECD-Fc fusion molecule, wildtype PD-L2 ED-Fcfusion molecule or variant ICOSL Fc-fusion molecules were bound to thereceptors in 4 point titrations. Each titration was globally fit tocalculate the associate (k_(on) and dissociation (K_(dis)) of eachprotein. Loading response of anti-human capture sensors of each receptorbeing tested with the variant ICOSL ECD-Fc fusion molecule wasdetermined. The dissociation constant (KD) was calculated and comparedto wildtype to determine a fold improvement value (fold imp.).

Binding results to ICOS are set forth in Table 19, to CD28 are set forthin Table 20 and to CTLA-4 are set forth in Table 21. The exemplary aminoacid substitutions depicted in Table 19-21 are designated by amino acidposition number corresponding to the respective reference unmodifiedICOSL ECD sequence set forth in SEQ ID NO:32.

TABLE 19 ICOS ForteBio Binding Assay SEQ ID NO KD K_(on) K_(dis) FullFold ICOSL mutation(s) (ECD) Response (M) (1/Ms) (1/s) R² Imp. WT ICOSL32 0.73 8.83E−10 1.78E+05 1.58E−04 0.9908 — N52H, I143T 135 0.873.32E−10 3.13E+05 1.04E−04 0.9683 2.7 N52H, N57Y, Q100R, C198R 365 0.744.92E−10 3.85E+05 1.89E−04 0.9882 1.8 N52H, N57Y, Q100R, V122A 290 0.674.72E−10 3.77E+05 1.78E−04 0.9775 1.9 N52H, N57Y, Q100R, F172S 291 0.684.20E−10 4.34E+05 1.82E−04 0.9545 2.1 N52Y/N57Y/F138L/L203P 112 0.647.69E−10 2.22E+05 1.71E−04 0.9782 1.1 V11E, N30D, N52H, N57Y, 308 0.673.62E−10 3.55E+05 1.29E−04 0.9687 2.4 H94E, L96I, L98F, N194D, V210A,I218T N52H, N57Y, Q100R, L102R, 367 0.76 4.77E−10 3.29E+05 1.57E−040.9616 1.9 V110D, H115R, C198R N52H, N57Y, Q100R 283 0.74 3.69E−102.87E+05 1.06E−04 0.9817 2.4 N52H, Q100R 285 0.79 3.73E−10 4.45E+051.66E−04 0.968 2.4 N52H, N57Y, Q100R, V110D, 364 0.60 1.29E−09 1.66E+052.15E−04 0.9846 0.7 C198R, S212G N52H, N57Y, Q100P 113 0.73 3.82E−103.71E+05 1.42E−04 0.9729 2.3 E16V, N52H, N57Y, Q100R, 300 0.75 5.43E−102.65E+05 1.44E−04 0.9848 1.6 V110D, H115R, Y152C, K156M, C198R N30D,K42E, N52S 324 0.80 3.71E−10 4.48E+05 1.66E−04 0.9651 2.4 N52S, F120S,I143V, I224V 325 0.80 3.11E−10 5.03E+05 1.56E−04 0.9673 2.8 N52S, E90A323 0.88 3.40E−10 4.85E+05 1.65E−04 0.9792 2.6 N52H, N57Y, V110A, C198R,317 0.68 4.77E−10 3.15E+05 1.50E−04 0.976 1.9 R221I N52S, N194D 366 0.883.37E−10 3.38E+05 1.14E−04 0.9723 2.6 N52D 111 0.87 3.38E−10 3.91E+051.32E−04 0.9792 2.6 Wildtype PD-L2 ED-Fc — 0.03

TABLE 20 CD28 ForteBio Binding Assay SEQ ID NO KD K_(on) K_(dis) FullFold ICOSL mutation(s) (ECD) Response (M) (1/Ms) (1/s) R² Imp. WT ICOSL32 0.33 1.39E−08 6.69E+04 9.29E−04 0.9715 — N52H, I143T 135 0.955.25E−10 4.27E+05 2.24E−04 0.9877 26.5 N52H, N57Y, Q100R, C198R 365 1.144.47E−10 4.12E+05 1.84E−04 0.9877 31.0 N52H, N57Y, Q100R, V122A 290 1.043.90E−10 4.07E+05 1.59E−04 0.9878 35.6 N52H, N57Y, Q100R, F172S 291 1.062.93E−10 4.26E+05 1.25E−04 0.9836 47.3 N52Y/N57Y/F138L/L203P 112 0.867.83E−10 1.79E+05 1.40E−04 0.993 17.7 V11E, N30D, N52H, N57Y, 308 0.925.53E−10 2.54E+05 1.40E−04 0.9906 25.1 H94E, L96I, L98F, N194D, V210A,I218T N52H, N57Y, Q100R, L102R, 367 1.10 3.66E−10 3.41E+05 1.25E−040.986 37.9 V110D, H115R, C198R N52H, N57Y, Q100R 283 1.04 3.68E−103.72E+05 1.37E−04 0.983 37.7 N52H, Q100R 285 1.09 4.01E−10 5.0SE+052.02E−04 0.9938 34.7 N52H, N57Y, Q100R, 364 0.94 8.96E−10 1.78E+051.60E−04 0.9961 15.5 V110D, C198R, S212G N52H, N57Y, Q100P 113 0.994.36E−10 3.29E+05 1.43E−04 0.9835 31.8 E16V, N52H, N57Y, Q100R, 300 1.065.03E−10 3.06E+05 1.54E−04 0.9872 27.6 V110D, H115R, Y152C, K156M, C198RN30D, K42E, N52S 324 0.54 1.95E−09 2.74E+05 5.33E−04 0.9772 7.1 N52S,F120S, I143V, I224V 325 0.84 9.10E−10 4.51E+05 4.10E−04 0.9742 15.3N52S, E90A 323 0.94 9.69E−10 4.74E+05 4.59E−04 0.978 14.3 N52H, N57Y,V110A, 317 0.94 5.63E−10 2.63E+05 1.48E−04 0.9781 24.7 C198R, R221IN52S, N194D 366 0.82 1.04E−09 3.53E+05 3.68E−04 0.9887 13.3 N52D 1110.86 1.16E−09 3.36E+05 3.90E−04 0.989 11.9 wildtype PD-L2 ED-Fc — −0.04

TABLE 21 CTLA-4 ForteBio Binding Assay SEQ ID NO KD K_(on) K_(dis) FullFold ICOSL mutation(s) (ECD) Response (M) (1/Ms) (1/s) R² Imp. WT ICOSL32 0.21 7.71E−08 1.92E+04 1.48E−03 0.8919 — N52H, I143T 135 0.966.78E−10 7.26E+05 4.92E−04 0.9641 113.8 N52H, N57Y, Q100R, C198R 3651.57 6.45E−10 4.79E+05 3.09E−04 0.9875 119.6 N52H, N57Y, Q100R, V122A290 1.43 5.76E−10 4.73E+05 2.72E−04 0.9926 133.9 N52H, N57Y, Q100R,F172S 291 1.47 5.36E−10 5.13E+05 2.75E−04 0.9924 144.0N52Y/N57Y/F138L/L203P 112 1.33 8.33E−10 3.45E+05 2.87E−04 0.9943 92.6V11E, N30D, N52H, N57Y, 308 1.50 6.48E−10 3.12E+05 2.02E−04 0.9943 119.0H94E, L96I, L98F, N194D, V210A, I218T N52H, N57Y, Q100R, L102R, 367 1.608.64E−10 4.79E+05 4.14E−04 0.9825 89.2 V110D, H115R, C198R N52H, N57Y,Q100R 283 1.65 7.19E−10 4.28E+05 3.08E−04 0.9895 107.2 N52H, Q100R 2851.17 5.92E−10 8.37E+05 4.96E−04 0.9629 130.3 N52H, N57Y, Q100R, 364 1.321.47E−09 2.34E+05 3.44E−04 0.9937 52.6 V110D, C198R, S212G N52H, N57Y,Q100P 113 1.51 6.47E−10 3.61E+05 2.33E−04 0.9911 119.2 E16V, N52H, N57Y,Q100R, 300 1.58 1.06E−09 4.24E+05 4.49E−04 0.9779 72.8 V110D, H115R,Y152C, K156M, C198R N30D, K42E, N52S 324 0.42 2.81E−09 2.42E+05 6.81E−040.9676 27.4 N52S, F120S, I143V, I224V 325 0.58 1.20E−09 3.10E+053.72E−04 0.9283 64.3 N52S, E90A 323 0.64 1.12E−09 3.28E+05 3.68E−040.9184 68.7 N52H, N57Y, V110A, C198R, 317 1.44 1.07E−09 4.0SE+054.32E−04 0.9811 72.3 R221I N52S, N194D 366 0.59 2.52E−09 2.66E+056.69E−04 0.9643 30.6 N52D 111 0.62 1.52E−09 4.16E+05 6.32E−04 0.923450.7 wildtype PD-L2 ED-Fc — 0.00

C. Coimmobilization Assay

Costimulatory bioactivity of ICOSL fusion variants was determined inanti-CD3 coimmobilization assays substantially as described in Example6. Approximately 0.37 nM, 1.3 nM or 10 nM mouse anti-human CD3 (OKT3,Biolegends, USA) was diluted in PBS with 10 nM or 40 nM variant ICOSLECD Fc or wild-type ICOSL ECD-Fc. This mixture was added to tissueculture treated flat bottom 96 well plates overnight to facilitateadherence of the stimulatory proteins to the wells of the plate. Thenext day, unbound protein was washed off the plates and 100,000 purifiedhuman pan T cells were added to each well. Cells were cultured 3 daysbefore harvesting culture supernatants and measuring human IFN-gammalevels with an ELISA kit.

Table 22 sets fort the amount of IFN-gamma (pg/mL) produced by cellsunder the various conditions in the anti-CD3 coimmobilization assay. Inthe Table, the amino acid substitutions of exemplary variant ECDICOSL-Fc fusions are designated by amino acid position numbercorresponding to the unmodified ICOSL ECD sequence set forth in SEQ IDNO: 32 and the corresponding SEQ ID NO identifier for the variant ECDfor each variant ICOSL ECD-Fc fusion molecule is set forth. The ratio ofIFN-gamma produced in the presence of each variant ICOSL ECD-Fc in thefunctional assay compared to in the presence of the correspondingunmodified (wildtype) ECD-Fc is shown (Fold ⬆WT). As shown,costimulatory signaling of the variant ICOSL-ECD-Fc molecules weresubstantially greater compared to wildtype ICOSL.

TABLE 22 Assessment of IFN-gamma Responses in Co-stimulation AssayIFN-gamma [pg/mL] SEQ 40 nM Ligand 10 nM Ligand 40 nM Ligand ID NO 10 mMFold 10 mM Fold 10 mM 1.1 nM 0.37 nM ICOSL mutation(s) (ECD) OKT3 ↑WTOKT3 ↑WT OKT3 OKT3 OKT3 E16V, N52H, N57Y, 300 14372 17.3 4903 29.98379.2 7422.8 2893.7 Q100R, V110D, H115R, Y152C, K156M, C198R N52H,N57Y, Q100R, 290 10640 12.8 6456 39.4 5636.2 4724.2 2246.3 VI22A N52H,N57Y, Q100R, 291 10379 12.5 3741 22.8 3979.7 4067.7 1415.5 F172S N52H,N57Y, Q100R, 365 9590 11.5 4048 24.7 4215.8 2787.1 1072.4 C198R N52H,N57Y, Q100R 283 9568 11.5 3270 19.9 4412.3 3862.0 1820.0 N52H, N57Y,Q100R, 367 6939 8.4 3234 19.7 5495.2 4081.6 1442.8 L102R, V110D, H115R,C198R N52S, F120S, I143V, 325 6567 7.9 717 4.4 2145.4 2185.7 646.1 I224VN52S, N194D 366 5690 6.8 272 1.7 2315.1 1485.0 1140.6 V11E, N30D, N52H,308 5345 6.4 1152 7.0 2747.0 3383.4 1701.2 N57Y, H94E, L96I, L98F,N194D, V210A, I218T N52S, E90A 323 5097 6.1 706 4.3 5019.8 3036.4 1482.4N52H, N57Y, V110A, 317 4737 5.7 520 3.2 2501.5 1632.1 937.5 C198R, R221IN52H, Q100R 285 4122 5.0 1466 8.9 5782.1 2861.4 967.5 N30D, K42E, N52S324 4080 4.9 273 1.7 1336.8 1260.7 541.1 N52H, N57Y, Q100P 113 3344 4.0229 1.4 2525.4 2439.5 1233.9 N52H, N57Y, Q100R, 364 3064 3.7 1471 9.02699.5 2629.9 678.2 V110D, C198R, S212G N52Y, N57Y, F138L, 112 2177 2.6200 1.2 1889.5 1757.9 808.8 L203P N52H, I143T 135 1906 2.3 138 0.81417.1 1367.9 275.2 WT ICOSL 32 831 1.0 164 1.0 558.8 377.7 152.0 N52D111 88 0.1 231 1.4 1288.9 1737.9 289.0

D. Mixed Lymphocyte Reaction for Assessment of Bioreactivity Suppression

Modulation of T cell activity by fusion variants was determined in amixed lymphocyte reaction (MLR) substantially as described in Example 6.Human monocytes were incubated for 6 days in the presence of IL-4 andGM-CSF and matured to dendritic cells with the additional of LPS for thefinal 24 hours. 1×10⁴ dendritic cells and 1×10⁵ human CFSE-labeled Tcells were plated per well and incubated for 4 days in the presence ofthree different concentrations (40 nM, 13.3 nM or 4.4 nM) of wildtype orrecombinant variant ICOSL ECD-Fc molecules diluted in PBS. The sameconcentrations of human IgG, PD-L2-Fc or Belatacept (CTLA4-Fc containingL104E and A29Y mutations) were used as controls. Supernatants wereharvested and IFN-gamma responses were characterized by ELISA.

FIG. 5 depicts the IFN-gamma production under the various conditions.The levels of IFN-gamma produced by cells in the presence of wild-typeICOSL is set forth by the horizontal line. No suppression of IFN-gammaproduction was observed in the presence of negative control proteinPD-L2-Fc. In contrast, most of the tested ICOSL variants exhibited somedegree of inhibition of IFN-gamma production in the MLR. Certainvariants exhibited substantial inhibition of IFN-gamma with very low tono detectable IFN-gamma produced in the cultures, even at the lowestconcentration of 4.4 nM tested. The percent MLR suppression in thepresence of 4.4 nM of variant of variant ECD ICOSL-Fc is set forth inTable 23. In the Table, the negative values indicate an inflammatoryeffect in the assay.

TABLE 23 Costimulatory bioactivity data for ICOSL in MLR SEQ ID % MLR NOSuppresion ICOSL mutation(s) (ECD) (4.4 nM) N52H, N57Y, Q100R, C198R 36593.6 N52H, N57Y, Q100R, V122A 290 94.4 N52H, N57Y, Q100R, F172S 291100.0 N52Y/N57Y/F138L/L203P 112 100.0 V11E, N30D, N52H, N57Y, 308 100.0H94E, L96I, L98F, N194D, V210A, I218T N52H, N57Y, Q100R, L102R, 367100.0 V110D, H115R, C198R N52H, N57Y, Q100R 283 98.2 N52H, Q100R 28597.5 N52H, N57Y, Q100R, V110D, 364 90.4 C198R, S212G N52H, N57Y, Q100P113 100.0 E16V, N52H, N57Y, Q100R, 300 100.0 V110D, H115R, Y152C, K156M,C198R N30D, K42E, N52S 324 −38.8 N52S, F120S, I143V, I224V 325 −44.2N52S, E90A 323 −30.4 N52H, N57Y, V110A, C198R, 317 100.0 R221I N52S,N194D 366 −22.3 N52H, I143T 135 −78.0 N52D 111 0.5

E. Assessment of Proliferation and Intracellular Cytokine Markers byFlow Cytometry

Carboxyfluorescein succinmidyl ester (CFSE) labeled pan T cells from MLRstudies as described above that had been incubated for 4 days in thepresence of wildtype or recombinant variant ICOSL ECD-Fc molecules werefurther tested for cytokine levels by restimulation with phorbolmyristate acetate (PMA)/Ionomycin for 6 hours in the presence of golgiinhibitor (Golgi/Block/Plug). T cells from the MLR study that had beenincubated with human IgG, anti-CD28, anti-ICOSL, PD-L2-Fc, or Belatacept(CTLA4-Fc containing L104E and A29Y mutations) also were restimulated. Tcells were stained for CD4 or CD8 surface markers, fixed, permeabilized,and intracellularly stained for various cytokines as set forth in Table24 and 25.

The percent (%) of CD4+ and CD8+ T cells that were positive for specificintracellular cytokines are shown in Table 24, respectively. The resultsshowed that a number of the variant ICOSL ECD-Fc molecules were able tosuppress one or more cytokines, including, in some cases, a majority ofcytokines. A total score and mean score are calculated to assess the sumeffects of individual molecules tested over the parameters examined inthis assay. Proliferation was also assessed and a percentage of cellsthat have divided as determined by CFSE dilution is also shown in Table24 and 25. Among the provided results, the results show that certainvariants show comparable or better activity than Belatacept,particularly from the CD8+ cells.

TABLE 24 Assessment of Proliferation and Intracellular Cytokine levelsof CD4+ T cells Variant SEQ % Cytokine+ ID NO (ECD) % % % % % % % %Total Mean or Protein Prolif IFNg+ IL4+ IL21+ IL22+ TNF+ IL2+ IL10+IL17A+ Score Score 308 3.0 9.9 3.0 1.0 1.3 34.9 31.3 0.1 0.2 41.0 4.6300 2.7 11.1 3.4 1.1 1.4 38.0 35.0 0.0 0.1 63.0 7.0 317 2.9 10.9 3.3 1.11.5 37.3 34.1 0.1 0.2 65.0 7.2 291 3.2 8.1 2.5 0.6 1.1 27.9 24.1 0.9 1.366.0 7.3 283 3.3 9.2 3.0 0.8 1.4 31.1 26.3 0.8 1.3 70.0 7.8 364 3.4 10.93.3 1.0 1.6 36.5 32.3 0.5 0.9 89.0 9.9 390 3.6 9.5 3.1 0.9 1.5 33.8 29.40.8 1.4 92.0 10.2 367 2.8 12.0 3.5 1.1 1.6 40.9 38.5 0.1 0.3 92.0 10.2CTLA-4-Ig: 10.7 10.5 2.7 2.4 2.0 24.5 19.6 0.6 1.4 99.0 11.0 L104E, A29Y(Belatacept) 112 3.5 12.0 3.8 1.3 1.6 41.3 36.7 0.2 0.4 109.0 12.1 2854.4 9.8 3.2 1.2 1.7 32.7 29.3 0.9 1.4 114.0 12.7 113 3.0 13.2 4.1 1.21.8 43.2 39.8 0.1 0.2 115.0 12.8 365 3.6 11.3 3.9 1.0 2.0 39.1 34.6 0.81.3 118.0 13.1 WT ICOSL 12.7 16.7 5.8 4.9 4.7 36.2 29.2 0.2 0.4 127.014.1 113 3.5 12.3 3.9 1.1 1.8 39.2 37.5 0.4 0.6 127.0 14.1 366 10.9 16.07.2 4.9 5.4 40.6 33.0 0.1 0.2 135.0 15.0 321 10.6 16.7 6.0 4.3 5.0 41.337.3 0.2 0.3 146.0 16.2 mlgG ctl 15.5 15.6 5.9 5.2 4.6 31.7 26.0 0.4 1.7146.0 16.2 PDL2 12.3 17.9 5.7 4.7 5.2 41.4 36.0 0.3 0.6 163.0 18.1 32311.9 17.7 6.2 5.2 5.3 42.4 37.5 0.2 0.4 167.0 18.6 WT ICOSL 12.8 17.46.3 5.4 5.6 38.9 32.1 0.3 0.6 167.0 18.6 Anti-ICOSL 15.5 16.3 6.5 5.25.5 35.1 29.1 0.7 2.0 168.0 18.7 135 12.6 17.4 6.5 5.4 4.9 44.3 37.2 0.40.5 179.0 19.9 HuIgG 12.7 17.1 6.3 5.9 4.9 41.1 32.4 0.7 1.3 181.0 20.1Anti-CD28 88.2 42.9 5.0 5.8 5.4 43.5 25.5 0.4 1.4 183.0 20.3 325 12.718.7 6.5 5.4 5.6 44.2 40.0 0.1 0.3 186.0 20.7 111 13.7 18.3 6.8 5.9 6.142.1 35.2 0.3 0.5 194.0 21.6

TABLE 25 Assessment of Proliferation andIntracellular Cytokine levels ofCD8+ T cells Variant SEQ % Cytokine+ ID NO (ECD) % % % % % % % % TotalMean or Protein Prolif IFNg+ IL4+ IL21+ IL22+ TNF+ IL2+ IL10+ IL17A+Score Score 308 4.2 8.4 1.6 2.2 47.2 7.6 8.0 0.2 0.1 67.0 7.4 300 3.88.5 2.0 2.2 47.2 8.1 9.1 0.1 0.0 69.0 7.7 317 3.9 8.8 2.0 1.7 45.6 8.49.0 0.1 0.1 64.0 7.1 291 3.8 7.0 1.4 1.8 46.6 5.9 5.9 1.1 1.1 78.0 8.7283 4.3 8.4 1.9 1.7 50.2 7.1 6.6 1.1 1.1 98.0 10.9 364 4.1 9.0 1.8 1.846.4 8.2 8.5 0.7 0.8 87.0 9.7 390 4.1 7.9 1.8 1.8 49.8 7.3 7.2 1.0 1.293.0 10.3 367 3.5 9.0 1.7 2.0 47.3 8.6 10.4 0.2 0.2 78.0 8.7 CTLA-4-Ig:12.3 14.5 2.0 3.2 38.1 11.2 8.8 0.9 1.6 121.0 13.4 L104E, A29Y(Belatacept) 112 4.2 9.6 1.9 1.8 40.4 9.4 9.9 0.3 0.3 81.0 9.0 285 5.49.5 2.0 2.7 44.2 7.8 8.4 1.2 1.3 112.0 12.4 113 3.7 9.5 1.9 1.3 44.4 9.410.5 0.1 0.1 62.0 6.9 365 4.1 9.6 2.3 1.8 46.8 9.0 9.3 0.9 1.0 122.013.6 ICOSL 17.2 22.3 4.9 6.4 46.4 22.0 15.7 0.4 0.7 181.0 20.1 113 4.29.5 2.0 2.1 45.6 8.9 10.7 0.5 0.5 110.0 12.2 366 14.5 19.4 5.6 4.8 48.419.2 13.8 0.1 0.2 142.0 15.8 321 13.4 18.9 4.3 5.0 46.3 18.4 15.4 0.30.6 138.0 15.3 mlgG ctl 20.2 25.0 4.4 4.1 35.5 24.6 15.8 0.7 1.8 174.019.3 PDL2 15.6 21.2 4.1 4.8 44.1 20.7 15.6 0.5 0.8 147.0 16.3 323 15.420.8 4.7 5.6 44.9 20.6 17.1 0.2 0.5 149.0 16.6 WT ICOSL 17.5 22.1 5.54.6 45.0 21.0 12.8 0.2 0.4 148.0 16.4 Anti-ICOSL 21.5 26.4 4.8 5.3 33.426.9 19.0 1.0 2.3 198.0 22.0 135 17.2 22.2 4.7 6.7 39.4 22.5 18.1 0.80.9 178.0 19.8 HuIgG 15.9 21.5 4.4 6.4 41.6 21.4 15.1 1.4 1.7 179.0 19.9Anti-CD28 60.6 44.3 3.5 2.1 38.6 32.5 16.0 1.2 1.6 182.0 20.2 325 16.522.0 4.9 5.4 44.0 21.9 18.5 0.2 0.6 161.0 17.9 111 17.7 22.8 5.3 6.145.4 22.9 16.7 0.4 0.6 183.0 20.3

Example 16 Assessment of Cytokine Production in B-T Cell Co-Culture

B cells and CD4+ T cells were purified from the same donor and labeledwith CSFE and plated in 96 well plates in 1:1 cellular ratios at 5×10⁴cells of each per well. Variant ICOSL ECD-Fc fusion molecules orBelatacept were added at a final concentration of 40 nM per well. Cellswere either unstimulated or incubated with 100 ng/mL of Staphylococcusenterotoxin B (SEB), 1 μg/mL of Pokeweed Mitogen (PWM) or both for 7days at 37° C. in a final volume of 200 μl/well.

Cells were harvested and surface stained for the following B and T celllineage markers (IgM, IgD, CD38, CD138, CD27, CD19, CD4, CD3).Proliferation was assessed by flow cytometry and culture supernatantswere analyzed for IL-5, IL-13 or IL-21 cytokines using a LEGENDplexhuman Th cytokine detection kit (Biolegend, USA).

As shown in FIG. 6A, the number of dividing B cells was reduced in B/Tcell co-cultures when incubated in the presence of variant ICOSL ECDFc-fusion molecules compared to no protein controls. The degree ofantagonistic effect for the tested exemplary variants was similar toCTLA-4-Ig Belatacept (L104E, A29Y). Likewise, as shown in FIGS. 6B-6D,variant ICOSL ECD Fc-fusion molecules inhibited cytokine production inprimary human B cell/T cell coculture in vitro compared to no proteincontrols as well as cultures containing wild-type ICOSL control.Compared to Belatacept, exemplary tested variant ICOSL ECD Fc-fusionmolecules were more effective in blocking cytokine production in somecases.

Example 17 Assessment of Survival and Disease Activity inGraft-Versus-Host-Disease (GvHD) Model

Exemplary ICOSL variant ECD-Fc protein, were assessed for activity in agraft-versus-host-disease (GvHD) model. Female NSG mice (n=10 per group)were irradiated (100 rad) and administered 10 mg of gamma globulinsubcutaneously on Day −1. On Day 0, the mice received 10 million humanperipheral blood mononuclear cells (PBMCs) and intraperitoneal injectiondosing of either 100 μg of WT-ICOSL ECD Fc, a variant ICOSL ECD Fcmolecule N52H/I143T (ECD set forth in SEQ ID NO: 135), a variant ICOSLECD Fc molecule N52H/N57Y/Q100P (ECD set forth in SEQ ID NO: 113), 75 μgof Belatacept (CTLA-4-Ig L104E/A29Y; U.S. Patent Application PublicationNumber US20160271218) or saline as control. On Day 15, engrafted humanCD45+ cells were phenotyped by flow cytometry. After the study wasterminated on Day 35, endpoint measurements of survival, body weightloss, and disease activity were evaluated.

FIG. 7A shows the survival of GVHD mice treated with saline, WTICOSL-ECD Fc, the variant ICOSL ECD-Fc molecules, or Belatacept. Asignificant difference in the survival of mice administered variantICOSL ECD-Fc N52H/N57Y/Q100P (ECD set forth in SEQ ID NO: 113) comparedto mice administered saline or WT ICSOL ECD-Fc was observed (p<0.0001 byMantel-Cox and Gehan-Breslow-Wilcoxon tests). FIG. 7B shows similardifferences between the body weight loss of mice treated with saline, WTICOSL ECD-Fc, the variant ICOSL ECD-Fc molecules, or Belatacept over thecourse of the study.

A disease activity index (DAI) was determined three times a week duringthe study and evaluated the scoring of weight loss, posture, activity,appearance of the hair coat and skin of the mice. The grade of diseaseover the course of the study is shown in FIG. 7C. Treatment groups thatreceived ICOSL Fc variant N52H/N57Y/Q100P (ECD set forth SEQ ID NO: 113)or Belatacept showed significantly improved DAI scores. The percentageof human T cells in the peripheral blood on day 14 of the study was alsoassessed by flow cytometry. Measurements were averaged by treatmentgroup and error bars represent standard error of the mean (SEM). FIG. 7Dshows the percent of live CD3+/CD4+ or CD3+/CD8+ cells in the blood.Treatment groups that received variant ICOSL ECD Fc with N52H/N57Y/Q100P(ECD set forth SEQ ID NO: 113) or Belatacept showed significantlydifferent levels of CD4+ T cells compared to saline treatment group(p=0.008 and 0.006, respectively, by unpaired t-test). This studydemonstrates the therapeutic effect of the variant ICOSL Fc variantprotein on human primary T cells and GVHD during in vivo modeling.

A similar study with additional variant ICOSL ECD-Fc fusion moleculeswas carried out including variant ICOSL ECD-Fc N52H/N57Y/Q100R/C198R(ECD set forth in SEQ ID NO: 365), N52H/N57Y/Q100R/F172S(ECD set forthin SEQ ID NO: 291), and N52H/N57Y/Q100P (ECD set forth in SEQ ID NO:288). N52H/Q100R (ECD set forth in SEQ ID NO: 285), which had reducedactivity in the in vitro MLR studies as shown above, also was tested.NSG mice (n=9/group) were treated as described above. In this study,dosing with the ICOSL-Fc or Belatacept continued 3 times per week fromday 0 through day 37 and surviving mice were terminated on day 49. Todetermine statistical differences in survival proportions among groups,data were analyzed using the Mantel-Cox (log-rank) test. The resultingsurvival curves are shown in FIG. 7E. Belatacept and ICOSL-Fc variantsN52H/N57Y/Q100R/C198R (ECD set forth in SEQ ID NO: 365),N52H/N57Y/Q100R/F172S (ECD set forth in SEQ ID NO: 291), andN52H/N57Y/Q100P (ECD set forth in SEQ ID NO: 288) significantlyprolonged survival as compared to saline and WT ICOSL-Fc (p<0.001). Themean DAI scores were plotted for the time course of the experiment, withthe last observation (i.e. mean scores collected on days of termination)carried forward on the graph for those groups terminated prior to thelast planned study day (Day 49). Significant differences among groupsfor data over time (i.e. DAI scores) were determined using 2-wayrepeated measures ANOVA for ‘treatment’ effects. The resulting DAIscores are shown in FIG. 7F. Belatacept and ICOSL-Fc variantsN52H/N57Y/Q100R/C198R (ECD set forth in SEQ ID NO: 365),N52H/N57Y/Q100R/F172S (ECD set forth in SEQ ID NO: 291), andN52H/N57Y/Q100P (ECD set forth in SEQ ID NO: 288) significantly reducedDAI scores as compared to saline and WT ICOSL-Fc (p<0.001), and betweenBelatacept and ICOSL-Fc variant with N52H/N57Y/Q100R/C198R by 2-wayANOVA for DAI scores (p=0.035).

The activity of the variant ICOSL-Fc, in some cases, was improvedcompared to treatment with belatacept. Administration of ICOSL-Fcvariants protected from effects of GVHD, as evidenced by enhancedsurvival and attenuated disease development, at levels comparable to orbetter than belatacept. The activity in this model correlated with invitro activity, since WT ICOSL-Fc and variant ICOSL-Fc with mutationsN52H/Q100R (ECD set forth in SEQ ID NO: 285) were not as effective inthis model.

Example 18 Assessment of Activation by Stacked Molecules

Stacked variant IgV Fc fusion proteins containing an NKp30 IgV domain asa localizing domain (designated “L”) and an ICOSL IgV domain as acostimulatory domain (designated “C”) were generated and assessedsubstantially as described in Example 8. Specifically, the constructstested in this experiment include: (1) a stacked construct with variantIgV Fc fusion protein (vIgD C-L) containing an NKp30 composed of Ig ofconsensus NKp30 variant (SEQ ID NO: 143) with the IgV domain of ICOSLvariant N52H/N57Y/Q100P (ECD set forth in SEQ ID NO: 113 and IgV setforth in SEQ ID NO:201); (2) a stacked construct with the NKp30 wildtypedomain Ig domain (SEQ ID NO:214) and V domain of wildtype ICOSL (WT C-L)(SEQ ID NO:196); (3) a construct with wild-type ICOSL IgV domain (WT Cdomain; SEQ ID NO:196), and (3) a construct with the wildtype NKp30domain (WT L domain; SEQ ID NO: 214).

CD32+K562 cells were engineered to stably express B7-H6 on the cellsurface. The cells were then treated with mitomycin and plated with panT-cells in the presence of absence of 10 nM anti-CD3 and stackedvariants or control domains at 100, 33, 11, or 3.7 nM. Cells werecultured 3 days before harvesting culture supernatants and measuringhuman IFN-gamma levels using an ELISA.

As shown in FIG. 8A, both variant and wildtype costimulatory-localizingdomain stacks were able to localize to the engineered K562 cells anddeliver a co-stimulatory signal to the pan-T-cells to induce IFN-gammasecretion. The stacked construct with variant IgV Fc fusion protein(vIgD C-L) showed increased functional activity results at allconcentrations tested, while the individual domain components had noeffect when not combined with each other.

Example 19 Assessment of Delayed Type Hypersensitivity In Vivo

Variant ICOSL ECD-Fc fusion molecules were assessed foranti-inflammatory activity in vivo in the mouse delayed-typehypersensitivity (DTH) model. Delayed-type hypersensitivity immunereactions were elicited in ovalbumin (OVA)-sensitized mice and responseafter challenge was assessed. The variant ICOSL ECD-Fc fusion moleculestested contained a variant ECD with the following amino acidsubstitutions: N52H/N57Y/Q100P (ECD set forth in SEQ ID NO: 113),N52H/Q100R (ECD set forth in SEQ ID NO: 285), or N52H/N57Y/Q100R/F172S(ECD set forth in SEQ ID NO: 291). The variants were fused to either anFc backbone containing mutations C220S/L234A/L235E/G237A by EU numbering(designated Fc #1) set forth in SEQ ID NO: 477) or an Fc backbonecontaining mutations C220S/E233P/L234V/L235A/G236del/S267K by EUnumbering (designated Fc #2) set forth in SEQ ID NO:478) either with orwithout a G4S (GGGGS; SEQ ID NO: 636) linker. Table 26 sets forth thetested constructs:

TABLE 26 ICOSL ECD-Fc Fusion Constructs ICOSL ECD Fc (SEQ ID NO) G4SLinker (SEQ ID NO) N52H/N57Y/Q100P 113 + 477 (G4S)-Fc #1 N52H/Q100R285 + 477 (G4S)-Fc #1 N52H/N57Y/Q100R/ 291 + 478 F172S (G4S)-FcN52H/N57Y/Q100R/ 291 + 477 F172S (G4S)-Fc N52H/N57Y/Q100R/ 291 − 477F172S-Fc

For sensitization, 8-week old female BALB/c mice were injectedsubcutaneously with 100 μg of OVA emulsified in Sigma Adjuvant (100 μL;catalog number 56322-1VL) at the base of the tail on day 0. On days 1and 4, the mice were administered variant ICOSL ECD-Fc fusion proteins,75 μg of CTLA-4 Fc (abatacept), or PBS as negative control byintraperitoneal injection. On day 7 at two to three hours prior to OVAchallenge, the mice were further administered PBS as a control, 75 μg ofCTLA-4 Fc (abatacept from Orencia), or the indicated variant ICOSLpolypeptide by intraperitoneal injection. Abatacept and variant ICOSL-Fcfusion molecules were dosed at molar equivalents.

For OVA challenge, an intradermal injection of 10 μg OVA in the left earpinnae in a 10 μL volume of PBS was delivered under gas isofluraneanesthesia 2-3 hours following therapeutic treatment. Baseline earthickness was measured prior to OVA challenge.

On day 8, ear thickness was measured under isoflurane anesthesia usingMitutoyo calipers and the change in ear thickness before and after OVAchallenge was determined. As shown in FIG. 9, mice treated with theindicated variant ICOSL ECD-Fc fusion molecules showed significantlyless OVA-induced ear swelling as compared to PBS control (<0.0001 by1-way ANOVA). There were no significant differences between earthickness for mice treated with abatacept compared to any of theindicated variant ICOSL ECD-Fc fusion molecules tested or between thevariant ICOSL treatments. These results demonstrate that variant ICOSLmolecules can reduce immune responses in vivo.

Example 20 Generation and Assessment of Binding and Activity of VariantICOSL IgSF Domain-Containing Molecules

Additional variant ICOSL IgSF (e.g. ECD) domain-containing moleculeswere generated, as described below. In each of the Tables below, theTable indicates amino acid substitutions in the ECD of the variant ICOSLas designated by amino acid position number corresponding to amino acidpositions in the respective reference (e.g., unmodified) ICOSLextracellular domain (ECD) sequence set forth in SEQ ID NO:32. In somecases, the removal of the AAA linker sequence of the variant ICOSLECD-Fc is indicated by “AAAA”. Column 2 sets forth the SEQ ID NOidentifier for each variant ECD domain contained in the variant ECD-Fcfusion molecule.

A. Generation of Additional Variants

1. Solubility Variants

From a collection of mutants containing the following mutationsincluding E16V, N30D, K42E, N52H, N52Y, N52S, N57Y, E90A, Q100R, Q100P,L102R, V110D, H115R, F120S, V122A, F138L, I143V, I143T, H152C, K156M,F172S, N194D, C198R, L203P, R221I, I224V, the mutations H115R, F172S andC198R were identified as mutations that may potentially enhance proteinsolubility or enhance protein expression (‘solubility mutations’). Thesethree mutations (H115R, F172S and C198R) were randomly introduced bysite directed mutagenesis into the same set of clones to generate acollection of derivatives that contain one or more of these solubilitymutations. Because site directed mutagenesis reaction was carried outwith pooled mutagenic oligos reacted with pooled parental clones in asingle reaction, some of the clones also contain some mutations fromother parental clones. The generated variants contained between 3 to 10different amino acid mutations in various combinations, as summarized inTable 27A.

TABLE 27A Exemplary variant ICOSL polypeptides ECD SEQ Mutation(s) ID NOWild-type 32 N52H/N57Y/Q100R/H115R/C198R 435 N52H/N57Y/Q100R/F172S/C198R436 N52H/N57Y/Q100R/H115R/F172S/C198R 437N52H/N57Y/Q100R/H115R/I143V/F172S/C198R 438N52H/N57Y/Q100R/L102R/H115R/F172S/C198R 439 N52H/V122A/F172S/C198R 440N52H/N57Y/Q100R/H115R/F172S/N194D 441 N52H/N57Y/H115R/F172S/C198R 442N52H/N57Y/Q100R/H115R/C198R 443 N52H/N57Y/H115R 444N52H/N57Y/Q100R/H115R 445 N52H/N57Y/Q100R/H115R/F172S/I224V 446N52H/N57Y/Q100R/H115R/F172S 447 N52H/N57Y/Q100R/F172S 448N52H/Q100R/H115R/I143T/F172S 449 N52H/N57Y/Q100P/H115R/F172S 450N52Y/N57Y/Q100P/F172S 451 E16V/N52H/N57Y/Q100R/V110D/H115R/C198R 452E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/ 453 K156M/F172S/C198RN52S/E90A/H115R 454 N30D/K42E N52S/H115R 455N30D/K42E/N52S/H115R/C198R/R221I 456 N30D/K42E/N52S/H115R/C198R 457N30D/K42E/N52S/H115R/F172S/N194D 458 N52S/H115R/F120S/I143V/C198R 459N52S/H115R/F172S/C198R 460 N52H/N57Y/Q100P/C198R 461 N52H/N57Y/Q100PH115R/F172S/C198R 462 N52H/N57Y/Q100P/F172S/C198R 463N52H/N57Y/Q100P/H115R 464 N52H/N57Y/Q100P/H115R/C198R 465N52H/Q100R/C198R 466 N52H/Q100R/H115R/F172S 467N52H/Q100R/H115X/F172S/C198R 468 N52H/Q100R/H115R/F172S/C198R 469N52H/N57Y/Q100R/F172S/C198R 470

2. Back Variants

Particular exemplary mutations including N52H, N52Y, N57Y, Q100R, Q100P,F138L, C198R, L203P identified in select variants described in Example 6were further combined in the ECD of the reference (e.g., wild-type)ICOSL with reference to positions set forth in SEQ ID NO:32 to generateadditional combination variants. The generated variants containedbetween 1 to 3 different amino acid mutations in various combinations asset forth in Table 27B.

TABLE 27B Exemplary variant ICOSL polypeptides ECD SEQ Mutation(s) ID NOWild-type 32 Q100R 427 F138L/L203P 428 N52Y/F138L/L203P 429N57Y/Q100R/C198R 430 N57Y/F138L/L203P 431 N52H 110 N57Y 121 N57Y/Q100P122 Q100R/F138L 432 L203P 433

3. Glycosylation Variants

Exemplary glycosylation mutations selected from N52H, N52Q, N84Q, N119Q,N155H, N155Q, N168Q, N207Q were combined in various permutations in theECD of the reference (e.g., wild-type) ICOSL with reference to positionsset forth in SEQ ID NO:32 to generate additional combination variants.The generated variants contained between 1 to 5 different amino acidmutations in various combinations as set forth in Table 27C. Mutationsdesignated with an “X” indicate either an N or Q at the indicatedposition.

TABLE 27C (glyc): Exemplary variant ICOSL polypeptides ECD SEQMutation(s) ID NO Wild-type 32 N84Q 387 N119Q 388 N168Q 389 N207Q 390N52Q/N207X 391 N168X/N207X 392 N52Q/N168Q 393 N84Q/N207Q 394 N155Q/N207Q395 N119Q/N168Q 396 N119Q/N207Q 397 N119Q/N155X 398 N52Q/N84Q 399N52Q/N119Q 400 N84Q/N119Q 401 N52Q/N84Q/N168Q 402 N52Q/N84Q/N207Q 403N84Q/N155Q/N168Q 404 N84Q/N168Q/N207Q 405 N84Q/N155H/N207Q 406N155Q/N168Q/N207Q 407 N119Q N155Q/N168Q 408 N119Q/N168Q/N207Q 409N84Q/N119Q/N207Q 410 N119Q/N155H/N207Q 411 N84Q/N119Q/N155Q 412N52Q/N119Q/N155Q 413 N52H/N84Q/N119Q 414 N52H/N84Q/N168X/N207X 415N52Q/N84Q/N155X/N168X 416 N52Q/N84Q/N119Q/N168Q 417N84Q/N119Q/N155Q/N168Q 418 N84Q/N155Q/N168Q/N207Q 419N84Q/N119Q/N155Q/N207Q 420 N52Q/N84Q/N119Q/N207Q 421N52Q/N84Q/N119Q/N155Q 422 N52Q/N84Q/N119Q/N155Q/N207Q 423N84Q/N119Q/N155Q/N168Q/N207Q 424

B. Binding to Cell-Expressed Counter Structures

The additional variants were formatted as Fc-fusion proteins asdescribed in Example 4. The variant Fc-fusion molecules were assessed inbinding studies to assess specificity and affinity of ICOSL domainvariant immunomodulatory proteins for cognate binding partners. Expi293cells transfected with cognate binding partners, human CD28, ICOS andCTLA4, were used in binding studies as described in Example 6. MFI wasdetermined for each transfectant and compared to the correspondingunmodified (parental) ECD-Fc.

Results for the binding for exemplary variant ICOSL ECD-Fc fusionmolecules are shown in Tables 28A-C. As shown in Table 28A-C, ICOSL IgSF(e.g. ECD) domain variants generated with the various combinations ofspecific mutations exhibited increased binding for at least one, and insome cases more than one, cognate counter structure ligand.

C. Bioactivity Characterization with Anti-CD3 Coimmobilization Assay

The costimulatory bioactivity of generated variant Fc-fusion moleculeswas also assessed in anti-CD3 coimmobilization assays as described inExample 6. Table 28A-C depicts the ratio of IFN-gamma produced by eachvariant ECD-Fc compared to the corresponding unmodified (wildtype) ICOSLECD-Fc in the assay. Mutations designated with an “X” indicate a Q orthe wildtype residue corresponding to the indicated position of SEQ IDNO: 32 at the indicated position. As shown, variant Fc-fusion moleculesgenerated exhibited improved activities to increase immunologicalactivity.

TABLE 28A Molecule sequences, binding data, and costimulatorybioactivity data of variant ICOSL ECD-Fc molecules containing selectmutations Coimmobilization Binding with anti-CD3 ICOS CD28 CTLA-4IFN-gamma SEQ ID MFI MFI MFI pg/mL NO (parental (parental (parental(parental ICOSL Mutations (ECD) ratio) ratio) ratio) ratio) N52H, N57Y,Q100R, F172S, C198R 436 118145 59651 178790 5059 (1.33) (29.60) (41.12)(37.90) N52H, N57Y, Q100R, H115R, 437 125341 51604 211000 8218 F172S,C198R (1.41) (25.60) (48.53) (61.57) N52Y, N57Y, Q100P, F172S 451 12128063663 174224 8123 (1.37) (31.59) (40.07) (60.86) E16V, N52H, N57Y,Q100R, V110D, 453 107819 68883 170080 8936 H115R, Y152C, K156M, F172S,(1.22) (34.18) (39.12) (66.95) C198R N52S, H115R, F120S, I143V, C198R459 116235 25582 22483 125 (1.31) (12.69) (5.17) (0.93) N52H, N57Y,Q100P, C198R 461 107164 56103 172319 1258 (1.21) (27.84) (39.63) (9.43)N52H, N57Y, Q100P, H115R, F172S, 462 120864 54586 176637 5507 C198R(1.36) (27.08) (40.63) (41.26) N52H, N57Y, Q100P, F172S, C198R 463117954 59376 151265 3884 (1.33) (29.46) (34.79) (29.10) N52H, N57Y,Q100P, H115R 464 126221 53321 178812 4154 (1.42) (26.46) (41.13) (31.13)N52H, N57Y, Q100P, H115R, C198R 465 137004 55454 148417 5069 (1.55)(27.51) (34.14) (37.98) N52H, Q100R, C198R 466 111428 58608 116111 3729(1.26) (29.08) (26.71) (27.94) N52H, Q100R, H115R, F172S 467 10553258287 106295 5294 (1.19) (28.92) (24.45) (39.67) N52H, Q100R, H115X,F172S, C198R 468 106555 73397 171815 6961 (1.20) (36.42) (39.52) (52.16)N52H, Q100R, H115R, F172S, C198R 469 114223 66686 157154 7592 (1.29)(33.09) (36.15) (56.88) N52H, N57Y, Q100R, F172S, C198R 470 99350 61292182288 9167 (1.12) (30.41) (41.93) (68.68) N52H, N57Y, Q100R, H115R,F172S, 437 114057 52011 146471 6545 C198R (1.29) (25.81) (33.69) (49.04)N52H, N57Y, Q100R, H115R, F172S 447 136143 66516 177376 8527 (1.54)(33.00) (40.80) (63.89) N52H, N57Y, Q100R, H115R, F172S, 437 13297059633 133247 5999 C198R (1.50) (29.59) (30.65) (44.95) Q100R 427 6206416740 29654 35 (8.31) (8.31) (8.31) (0.26) Q100R ΔAAA 427 1594 1653533457 87 (8.20) (8.20) (8.20) (0.65) F138L, L203P 428 53804 1510 2151 35(0.75) (0.75) (0.75) (0.26) F138L, L203P ΔAAA 428 53044 1882 1623 35(0.93) (0.93) (0.93) (0.26) N52Y, F138L, L203P 429 99761 47369 673001489 (23.50) (23.50) (23.50) (11.16) N52Y, F138L, L203P ΔAAA 429 5957652865 66553 997 (26.23) (26.23) (26.23) (7.47) N57Y, Q100R, C198R 43058706 57739 99426 9962 (28.65) (28.65) (28.65) (74.64) N57Y, Q100R,C198R ΔAAA 430 98514 57694 131458 6763 (28.63) (28.63) (28.63) (50.67)N57Y, F138L, L203P 431 109472 42276 64477 4979 (20.98) (20.98) (20.98)(37.30) N57Y, F138L, L203P ΔAAA 431 97777 44924 64742 6507 (22.29)(22.29) (22.29) (48.75) N52H 110 91598 58264 103025 3393 (28.91) (28.91)(28.91) (25.42) N57Y 121 109031 43754 50683 4881 (21.71) (21.71) (21.71)(36.57) N57Y, Q100P 122 72480 60161 109522 2797 (29.85) (29.85) (29.85)(20.95) Q100R, F138L 432 65974 4485 8136 685 (2.23) (2.23) (2.23) (5.13)L203P 433 61554 1533 2031 2434 (0.76) (0.76) (0.76) (18.24) WildtypeICOSL ECD 32 88625 2015 4348 133 (1.00) (1.00) (1.00) (1.00)

TABLE 28B Molecule sequences, binding data, and costimulatorybioactivity data of variant ICOSL ECD-Fc molecules containing selectmutations Coimmobilization Binding with anti-CD3 ICOS CD28 CTLA-4IFN-gamma SEQ MFI MFI MFI pg/mL ID NO (parental (parental (parental(parental ICOSL Mutations (ECD) ratio) ratio) ratio) ratio) N52H, N57Y,Q100R, H115R 445 165027 51666 287581 5858 (1.97) (9.89) (60.27) (20.36)N52H, N57Y, Q100R, F172S 448 184449 51394 182109 3449 (2.20) (9.84)(38.16) (11.99) N52H, N57Y, Q100R, H115R, 446 165120 46636 274026 2053F172S, I224V (1.97) (8.93) (57.43) (7.13) N52H, N57Y, Q100R, H115R, 447164750 40046 259351 3722 F172S (1.97) (7.67) (54.35) (12.93) N52H, N57Y,Q100R, H115R, 435 186017 39073 200505 3909 C198R (2.22) (7.48) (42.02)(13.58) N52H, N57Y, Q100R, F172S, 436 181118 38233 210709 1199 C198R(2.16) (7.32) (44.16) (4.17) N52H, N57Y, Q100R, H115R, 437 155392 28828169736 3449 F172S, C198R (1.85) (5.52) (35.57) (11.99) N52H, N57Y,Q100R, H115R, 438 139977 31459 179089 1620 I143V, F172S, C198R (1.67)(6.02) (37.53) (5.63) N52H, N57Y, Q100R, L102R 439 146799 29636 2000002712 H115R, F172S, C198R (1.75) (5.68) (41.91) (9.43) N52H, N57Y, Q100R,H115R 441 150863 31304 167783 15607 F172S, N194D (1.80) (5.99) (35.16)(54.24) N52H, N57Y, H115R, F172S, 442 126909 35803 152858 5374 C198R(1.51) (6.86) (32.03) (18.67) N52H, N57Y, Q100R, H115R, 443 131730 37595139041 9306 C198R (1.57) (7.20) (29.14) (32.34) N52H, N57Y, H115R 444162632 49847 266878 2918 (1.94) (9.55) (55.93) (10.14) N52H, Q100R,H115R, I143T 449 132873 52058 186366 3086 F172S (1.59) (9.97) (39.06)(10.72) N52H, N57Y, Q100P, H115R, 450 148160 46851 246636 4987 F172S(1.77) (8.97) (51.69) (17.33) E16V, N52H, N57Y, Q100R, 452 154036 48674212905 5095 V110D, H115R, C198R (1.84) (9.32) (44.62) (17.71) N52S,E90A, H115R 454 142963 3597 3772 2241 (1.71) (0.69) (0.79) (7.79) N30D,K42E, N52S, H115R, 456 124095 8066 7751 417 C198R R221I (1.48) (1.54)(1.62) (1.45) N30D, K42E, N52S, H115R, 457 161734 2791 2919 841 C198R(1.93) (0.53) (0.61) (2.92) N30D, K42E, N52S, H115R, 458 117880 43954941 2904 F172S, N194D (1.41) (0.84) (1.04) (10.09) N30D, K42E, N52S,H115R, 455 114107 2935 2748 549 (1.36) (0.56) (0.58) (1.91) N52S, E90A,H115R, 454 120450 12768 23282 2890 (1.44) (2.45) (4.88) (10.04) N30D,K42E, N52S, H115R 455 115273 11964 22779 2241 (1.38) (2.29) (4.77)(7.79) N52S, H15R, F172S, C198R 460 95537 7614 21701 1458 (1.14) (1.46)(4.55) (5.07) Wildtype 32 83813 5222 4772 288 (1.00) (1.00) (1.00)(1.00)

TABLE 28C Molecule sequences, binding data, and costimulatorybioactivity data of variant ICOSL ECD-Fc molecules containingglycosylation mutations Coimmobilization Binding with anti-CD3 ICOS CD28CTLA-4 IFN-gamma SSEQ MFI MFI MFI pg/mL ID NO (parental (parental(parental (parental ICOSL Mutation(s) (ECD) ratio) ratio) ratio) ratio)N84Q 387 34426 1755 5757 100 (0.94) (1.16) (1.51) (2.03) N119Q 388 308064102 19836 81 (0.84) (2.70) (5.21) (1.66) N168Q 389 27041 1410 18641 67(0.74) (0.93) (4.90) (1.36) N207Q 390 36516 11923 25701 206 (1.00)(7.86) (6.76) (4.20) N52Q, N207X 391 30216 12086 27952 77 (0.83) (7.97)(7.35) (1.56) N168X, N207X 392 37191 5787 12280 104 (1.02) (3.81) (3.23)(2.12) N52Q, N168Q 393 32576 12638 27167 101 (0.89) (8.33) (7.14) (2.06)N84Q, N207Q 394 37176 5292 3153 31 (1.02) (3.49) (0.83) (0.63) N155Q,N207Q 395 34884 1489 987 73 (0.95) (0.98) (0.26) (1.48) N119Q, N168Q 39629099 2534 11289 51 (0.80) (1.67) (2.97) (1.05) N119Q, N207Q 397 326031861 6795 153 (0.89) (1.23) (1.79) (3.12) N119Q, N155X 398 38516 1531827498 173 (1.05) (10.10) (7.23) (3.52) N52Q, N84Q 399 33988 1675 3525 39(0.93) (1.10) (0.93) (0.80) N52Q, N119Q 400 35729 11040 26139 51 (0.98)(7.28) (6.87) (1.03) N84Q, NH9Q 401 34777 1493 2877 39 (0.95) (0.98)(0.76) (0.80) N52Q, N84Q, N168Q 402 27021 1584 958 38 (0.74) (1.04)(0.25) (0.78) N52Q, N84Q, N207Q 403 39942 13396 26360 37 (1.09) (8.83)(6.93) (0.76) N84Q, N155Q, N168Q 404 27812 357 466 30 (0.76) (0.24)(0.12) (0.61) N84Q, N168Q, N207Q 405 30659 737 861 25 (0.84) (0.49)(0.23) (0.52) N84Q, N155H, N207Q 406 13557 685 607 29 (0.37) (0.45)(0.16) (0.59) N155Q, NI68Q, N207Q 407 13999 277 317 40 (0.38) (0.18)(0.08) (0.82) N119Q, N155Q, N168Q 408 36896 4094 2179 50 (1.01) (2.70)(0.57) (1.02) N119Q, N168Q, N207Q 409 29543 921 3744 72 (0.81) (0.61)(0.98) (1.47) N84Q, N119Q, N207Q 410 21357 569 640 59 (0.58) (0.38)(0.17) (1.20) N119Q, N155H, N207Q 411 37310 614 931 86 (1.02) (0.40)(0.24) (1.75) N84Q, N119Q, N155Q 412 2675 262 291 34 (0.07) (0.17)(0.08) (0.70) N52Q, N119Q, N155Q 413 27853 552 772 42 (0.76) (0.36)(0.20) (0.87) N52H, N84Q, N119Q 414 40700 4580 4601 39 (1.11) (3.02)(1.21) (0.80) N52H, N84Q, N168X, N207X 415 8796 587 481 32 (0.24) (0.39)(0.13) (0.66) N52Q, N84Q, N155X, N168X 416 43521 6605 4811 32 (1.19)(4.35) (1.26) (0.66) N52Q, N84Q, N119Q, N168Q 417 39342 4519 3300 37(1.07) (2.98) (0.87) (0.76) N52Q, N84Q, N119Q, N207Q 421 7011 602 433 37(0.19) (0.40) (0.11) (0.75) Wildtype ICOSL ECD 32 36602 1517 3804 49(1.00) (1.00) (1.00) (1.00)

Example 21 Generation and Assessment of Fusion Molecules withHER2-Targeting Antibody

This Example describes the generation and assessment of variant ICOSLECD-Fc fusion molecules conjugated with a tumor targeting agent to forma conjugate (“vIgD conjugate”).

The V-domain only of the ICOSL vIgD (N52H/N57Y/Q100P; set forth in SEQID NO: 201) was fused to the amino and carboxyl termini of the lightchain (FIG. 10A) and heavy chain (FIG. 10B) of a HER2 targeting antibodywith intervening GGGSGGGS linkers. Exemplary configurations of vIgDconjugates are shown in FIG. 10C.

To assess HER2 binding, HER2 DNA or mock Expi293 transfectants werestained with titrated amounts of a HER2 targeting antibody containingthe variant ICOSL conjugate (vIgD N52H/N57Y/Q100P conjugate) at aconcentration of 100 pM to 100 nM. Control proteins, including wildtypeICOSL ECD-Fc fusion, wildtype PD-L2 IgV-Fc fusion, and variant ICOSLECD-Fc fusion molecule with mutations at N52H/N57Y/Q100P, were alsotested. Mean Fluorescence Intensity (MFI) or percent positive cells wasdetermined for each transfectant as described in Example 6. All IgSFconjugates generated as shown in FIG. 11A-11B retained binding to HER2compared to the endogenous level of HER2 expression observed in Expi293cells. Similarly, vIgD conjugates also showed binding to cognate bindingpartners of ICOSL including CD28, CTLA-4, and ICOS.

Protein bioactivity and proliferation of human primary T cell in vitroassays were also characterized as described in Example 6. vIgDconjugates were bound overnight to 96-well plates at 30-0.1 nM in thepresence of 10 nM anti-CD3. The plates were washed and 100,000CFSE-labeled pan T cells were added to the plates and incubated for 72hours. IFN gamma levels in supernatant were assayed by ELISA. As shownin FIG. 12, vIgD conjugates with the indicated configurations showedgreater IFN gamma secretion and proliferation compared to parentalwild-type ICOSL ECD-Fc fusion molecule conjugate.

Example 22 Nanostring Transcriptional Signature of Primary Human T Cells

Tissue culture plates were coated with 10 nM anti-CD3 with 40 nM of anFc-control protein, wild-type ICOSL-Fc, wild-type CD80-Fc, both of theseproteins, or variant ICOSL Fc-fusion proteins with mutations asindicated. Purified human T cells were then plated on the protein coatedplates and incubated at 37° C. Cultures from each treatment groupdescribed above were harvested at 24, 48 and 72 hours and total RNA wasprepared from each cell sample. The RNA was transferred to Nanostringand a Cancer Immune chip was used to quantitate transcripts of 750 genein each sample. Transcript values were normalized using Nanostring'sproprietary software allowing comparison of transcript levels betweentreatment groups and over the various time points. As shown in FIG. 18and FIG. 19, the variant ICOSL ECD-Fc polypeptides tested show alteredinflammatory activity compared to wildtype CD80 ECD-Fc, wildtype ICOSLECD-Fc, or a combination of both.

Example 23 Generation and Assessment of Fusion Molecules withHER2-Targeting Antibody

Proliferation of human T-cells co-cultured with VmAbs and HER2expressing target cells was also characterized. CFSE-labeled pan T-cellswere stimulated for 72 hours with K562-derived artificial target cellsdisplaying cell surface anti-CD3 single chain Fv (OKT3) and HER2 in thepresence of VmAbs or control proteins. Proliferation was measured byflow cytometric analysis of CFSE-dilution on CD4⁺ or CD8⁺ stainedT-cells. Vmabs were assayed varying either target cell number or theconcentration of the VmAb utilized. In the first assay, K562 targetcells were titrated from 2500 to 78 cells/well and added to 100,000T-cells for an effector:target (E:T) range of 40 to 1280:1. VmAbs,parental IgSF domain, or WT ICOSL were added at 1000 pM. In the secondassay, K562 target cells were added at 625 cells/well to 100,000 T-cellsfor an effector:target ratio of 160:1. VmAbs or control proteins weretitrated and added at 3000 to 37 pM. As shown in FIGS. 20A and 20B, bothconfigurations of the assay demonstrate VmAbs containing thevIgD-conjugate provide superior proliferation compared to the parentalantibody, parental IgSF domain, or WT ICOSL. Additionally,vIgD-conjugates mediated proliferation at low E:T ratios (1280:1) or atlow protein concentrations (37 pM).

Example 24 Generation and Assessment of Engineered Cells Expressing aTransmembrane Immunomodulatory Protein and a T Cell Receptor

This Example describes the expression of various variant ICOSL IgSFdomain-containing transmembrane immunomodulatory proteins (TIPs) with anexemplary recombinant E6-specific T cell receptor (TCR) in human T cellsand assessment of T cell proliferation.

Human HLA-A2+ T cells were activated on Day 0 with anti-CD3/anti-CD28activation beads (ThermoFisher Scientific, USA) and transduced, on day1, with a TCR specific to HPV E6 (described in WO 2015/009606) andvarious transmembrane immunomodulatory proteins (TIPs) containing avariant ICOSL IgSF domain. The exemplary ICOSL-TIPs had anaffinity-modified IgSF domain containing amino acid mutationscorresponding to eitherE16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R or N52H/N57Y/Q100Rwith reference to positions in the ICOSL extracellular domain set forthin SEQ ID NO: 32. The ICOSL TIPs also included a transmembrane andcytoplasmic domain corresponding to residues 257-302 of SEQ ID NO: 5.For comparison, T cells also were co-transduced with the HPV E6 TCR andeither a WT CD80-TIP (set forth as amino acids 35-288 of SEQ ID NO: 1and encoded by the sequence of nucleotides set forth in SEQ ID NO:251)or a WT ICOSL-TIP (set forth as amino acids 19-302 of SEQ ID NO:5 andencoded by the sequence of nucleotides set forth in SEQ ID NO:252). Fortransduction, cells were transduced with a viral vector construct inwhich was inserted a polynucleotide encoding the TIP and the TCRα andTCRβ chain sequences, each separated from each other by a sequenceencoding a P2A ribosome skip sequence (SEQ ID NO: 863), forco-expression of the TIP and the TCR containing the TCRα and TCRβ chainin the engineered cells. Specifically, the nucleic acid construct hadthe following structure: ICOSL-P2A1-TCRβ-P2A2-TCRα, in which thenucleotide sequence P2A1 and P2A2 each encoded the P2A set forth in SEQID NO: 863 but differed in the nucleotide sequence to avoidrecombination between sequences.

As a control, T cells were mock transduced or transduced with theexemplary E6 TCR only.

The T cell activation beads were removed on day 3 and cytokines IL-2,IL-7, and IL-15 were added to the culture. On day 6 after transduction,cell surface expression of the TIP and TCR was assessed by flowcytometry, with 35-65% of engineered cells double positive for the TCRand TIP. The TCR-expressing cells were expanded in the presence of HPVE6 peptide to result in a population of cells that were >90% doublepositive for the TCR/TIP as assessed at day 14. On day 14, theengineered cells were incubated with HPV-infected cells, either asquamous cell carcinoma cell line UPCI:SCC152 (ATCC® CRL-3240™; HPV+,HLA-A2+), epidermoid carcinoma CaSki cells (ATCC® No. CRL-1550™; HPV+,HLA-A2+), or squamous cell carcinoma SiHa cells (ATCC® HTB-35™; HPV+,HLA-A2-). Proliferation of the engineered cells was assessed on day 3after initiation of co-culture with target cells. As shown in FIG. 21,increased proliferation of T cells engineered with E6 TCR was observedin the two HPV+ cell lines, but not significantly in the HPV-SiHa line.Engineered cells that co-expressed variant ICOSL TIPS had increasedproliferation in response to the HLA-A2+HPV+ target SCC152 and Caskicell lines.

Example 25 Generation and Assessment of Fc-Fusion ImmunomodulatoryProteins

Variant ICOSL IgSF (e.g. ECD) domain-containing molecules were formattedas Fc-fusion proteins substantially as described in Example 4, exceptusing various linkers and Fc molecules. To generate immunomodulatoryproteins that are Fc fusion proteins containing an ECD of ICOSL with atleast one affinity-modified domain (e.g. variant ICOSL ECD-Fc), theencoding nucleic acid molecule was generated to encode a proteindesigned as follows: variant (mutant) ECD linked directly or indirectlyvia a linker to an inert human IgG1 Fc. Specifically, the generatedimmunomodulatory proteins either did not contain a linker (none) orcontained a AAA or a G4S (SEQ ID NO: 636) linker. The inert human IgG1Fc contained mutations, by EU numbering, as follows:C220S/R292C/N297G/V302C (SEQ ID NO: 476),C220S/E233P/L234V/L235A/G236del/S267K (SEQ ID NO: 478),C220S/L234A/L235E/G237A (SEQ ID NO: 477), or allotypes thereof. Thereplacement C220S was included because the resulting proteins do notinclude an antibody light chain that can form a covalent bond with acysteine. The recombinant variant Fc fusion proteins were produced in293 cells and purified with Protein A substantially as described inExample 5.

The variant ICOSL Fc-fusion immunomodulatory proteins were assessed inbinding studies to assess binding for cognate binding partners. Expi293cells transfected with cognate binding partners, human CD28, ICOS andCTLA4, were used as target cells in binding studies as described inExample 6. MFI of binding of variant ICOSL Fc fusion immunomodulatoryproteins for target cell expressing each binding partner was determinedand compared to the binding of the corresponding unmodified (wildtype)ICOSL ECD-Fc to the same target cells. Modulation of T cell activity bythe variant ICOSL Fc-fusion immunomodulatory proteins was alsodetermined using a mixed lymphocyte reaction (MLR) substantially asdescribed in Example 6.

Results for the binding of exemplary variant ICOSL ECD-Fc fusionimmunomodulatory proteins containing various linkers and Fc regions areshown in Table 29. The Table indicates amino acid substitutions in theECD of the variant ICOSL as designated by amino acid position numbercorresponding to amino acid positions in the respective reference (e.g.,unmodified) ICOSL extracellular domain (ECD) sequence set forth in SEQID NO:32. Column 1 also sets forth the SEQ ID NO identifier for eachvariant ECD domain contained in the variant ICOSL Fc fusion. Column 2indicates the linker used in the Fc fusion protein and the SEQ ID NOidentifier for the linker. Column 3 sets forth the mutations in the Fcby EU numbering and the SEQ ID NO identifier for the Fc contained in thevariant ICOSL Fc fusion protein.

As shown in Table 29, similar results were observed for binding tocognate binding partners among the tested variant ICOSL Fc fusionproteins. These results indicate that the format of the Fc fusion withdifferent Fc molecules or different linkers did not impact the bindingof the ICOSL IgSF domain variants for their cognate binding partner. Inaddition, all Fc fusion formats, when provided as bivalent Fc moleculesin solution in an MLR reaction, exhibited an antagonist activity todecrease T cell activation compared to the reference (e.g. unmodified orwildtype) ECD-Fc molecule not containing the amino acid substitution. Insome cases, no detectable IFN-gamma was measured in the supernatantconsistent with complete blocking of interactions of costimulatoryligand cognate binding partners with their ligands to induce IFN-gammasecretion.

TABLE 29 Molecule sequences, binding data, and costimulatory bioactivitydata of variant ICOSL ECD-Fc molecules MLR Binding IFN- Linker CD28CTLA-4 ICOS gamma ICOSL Mutation (s) (SEQ Fc Mutations MFI MFI MFI pg/mL(SEQ ID NO) ID NO) (SEQ ID NO) (ΔWT) (ΔWT) (ΔWT) (ΔWT)N52H/N57Y/Q100R/F172S AAA C220S/R292C/N297G/V302C 67870 (13.0) 213333(22.7) 120042 (1.5) 3 (0.02) (291) (476) N52H/N57Y/Q100R/F172S AAAC220S/E233P/L234V/L235A/ 57272 (10.9) 192595 (20.5) 103983 (1.3) 0(0.00) (291) G236del/S267K (478) N52H/N57Y/Q100R/F172S AAAC220S/L234A/L235E/G237A 65506 (12.5) 193704 (20.6) 105432 (1.3) 0 (0.00)(291) (477) N52H/N57Y/Q100R/F172S G4S C220S/E233P/L234V/L235A/ (291)(636) G236del/S267K (478) N52H/N57Y/Q100R/F172S G4SC220S/L234A/L235E/G237A 67596 (12.9) 212875 (22.7) 106576 (1.4) 0 (0.00)(291) (636) (477) N52H/N57Y/Q100R/F172S none C220S/E233P/L234V/L235A/ —— — — (291) G236del/S267K (478) N52H/N57Y/Q100R/F172S noneC220S/L234A/L235E/G237A 59987 (11.5) 210061 (22.4) 106405 (1.4) 0 (0.00)(291) (477) N52H/Q100R (285) AAA C220S/R292C/N297G/V302C 57419 (11.0)190012 (20.2) 86522 (1.1) 30 (0.26) (476) N52H/Q100R (285) AAAC220S/E233P/L234V/L235A/ G236del/S267K (478) N52H/Q100R (285) AAAC220S/L234A/L235E/G237A 58772 (11.2) 211494 (22.5) 88969 (1.1) 25 (0.22)(477) N52H/Q100R (285) G4S C220S/E233P/L234V/L235A/ 62331 (11.9) 207285(22.1) 110512 (1.4) 31 (0.28) (636) G236del/S267K (478) N52H/Q100R (285)G4S C220S/L234A/L235E/G237A 70142 (13.4) 187699 (20.0) 125505 (1.6) 49(0.44) (636) (477) N52H/Q100R (285) none C220S/E233P/L234V/L235A/ 58726(11.2) 206110 (21.9) 110721 (1.4) 54 (0.48) G236del/S267K (478)N52H/Q100R (285) none C220S/L234A/L235E/G237A 62746 (12.0) 198281 (21.1)96948 (1.2) 16 (0.14) (477) N52H/N57Y/Q100P (113) AAAC220S/R292C/N297G/V302C 79792 (15.3) 193633 (20.6) 91384 (1.2) 1 (0.01)(476) N52H/N57Y/Q100P (113) AAA C220S/E233P/L234V/L235A/ 69603 (13.3)314593 (33.5) 103387 (1.3) 0 (0.00) G236del/S267K (478) N52H/N57Y/Q100P(113) AAA C220S/L234A/L235E/G237A 68729 (13.1) 171223 (18.2) 97068 (1.2)0 (0.00) (477) N52H/N57Y/Q100P (113) G4S C220S/E233P/L234V/L235A/ 67753(13.0) 188192 (20.0) 93424 (1.2) 1 (0.01) (636) G236del/S267K (478)N52H/N57Y/Q100P (113) G4S C220S/L234A/L235E/G237A 69887 (13.4) 160705(17.1) 104124 (1.3) 0 (0.00) (636) (477) N52H/N57Y/Q100P (113) NoneC220S/E233P/L234V/L235A/ 68979 (13.2) 184726 (19.7) 98512 (1.3) 0 (0.00)G236del/S267K (478) N52H/N57Y/Q100P (113) none C220S/L234A/L235E/G237A67863 (13.0) 154563 (16.5) 97714 (1.2) 0 (0.00) (477) WT (32) AAAC220S/R292C/N297G/V302C 5232 (1.0) 9394 (1.0) 78795 (1.0) 113 (1.00)(476)

Example 26 Expression of Variant ICOSL Molecules in CHO Cells

As an alternative to expressing variant ICOSL Fc fusion proteins inExpi293 cells as described in Example 5, suspension Chinese hamsterovary cells (ExpiCHO-S) cells were used to produce various ICOSLmolecules. A DNA construct encoding the exemplary variant ICOSL IgSF(e.g. ECD) Fc-fusion proteins containing the variant (mutant) ECDN52H/N57Y/Q100R/F172S (SEQ ID NO:291) linked to an inert Fc containingmutations C220S/L234A/L235E/G237A by EU numbering set forth in SEQ IDNO: 477 or an allotype thereof set forth in SEQ ID NO: 637, with aGSGGGGS linker (SEQ ID NO: 635) was used to transfect cells.

ExpiCHO-S cells, and reagents for transfection using the ExpiCHO™Expression System, were purchased from ThermoFisher Scientific (Cat #A29133). The cells were thawed and expanded per manufacture'srecommended protocol. After at least 2 passages, the cells were split 24hours pre-transfection and allowed to expand to high density. Cells werethen diluted to the number of cells for transfection, DNA complex wasformed with the ExpiFectamine™ CHO reagent and added to the cells. Oneday post DNA complex addition the ExpiCHO™ feed and ExpiFectamine™ CHOEnhancer were added to the culture, which was then placed in a 32 degreeC. incubator. The cell viability and cell mass were monitored and theculture was harvested when the viability fell below 80%. The culture wasthen centrifuged at low speed to remove the cell pellet, and the clearedsupernatant was 0.2 um sterile filtered. Protein was purified asdescribed in Example 5.

A. Protein Analysis

The purified variant ICOSL Fc fusion protein was run on SDS-PAGE andanalyzed by protein staining. Multiple bands were observed in cellsproduced from CHO cells but not from 293 cells, which is consistent withan observation that proteolysis clipping of the ICOSL was occurring whenexpressed in CHO cells. Table 30A depicts the molecular weight ofintact, single-clipped, and double clipped proteins calculated based onamino acid sequences and potential carbohydrates as observed bySDS-PAGE. Proteolysis of the ICOSL Fc-fusion proteins expressed inExpiCHO-S derived cells was observed, as indicated by the presence ofboth reduced/non-reduced clipped species with lower molecular weight(single and double clipping). Based on the size of the observed bandsand Mass Sprectromety analysis, these results are consistent with apotential cleavage site in ICOSL ECD corresponding to the sequenceLQQN/LT (“/” indicates potential cleavage site), thereby resulting incleavage before the stalk region of the ECD and removal of the Fcportion of the sequence in one or both chains of the Fc fusion protein.The observed protease cleavage may result in a heterogenous proteinproduct when produced in CHO cells. Also, for formats expressed astransmembrane immunomodulatory proteins, protease cleavage, occurring incertain cells, could lead to release of soluble protein from cells,thereby reducing cell surface-expressed forms of the variant protein onengineered cells.

TABLE 30A Reduced/Non-Reduced Species Detected After Capture and Elutionfrom Protein A Column Chromatography Using SDS-PAGE to Assay ProteolysisCalculated MW Observed (Apparent) MW¹ Non Non Reduced Reduced ReducedReduced Intact 105.6 kD aa 52.8 kD aa 150 kD 80 kD ~29 kD carb ~14.5 kDcarb 135 kD total 67.3 kD total Single 78.8 kD aa 52.8 kD aa 100 kD 80kD Clip ~16.5 kD carb ~14.5 kD carb 37 kD 95.3 kD total 67.3 kD total 26kD aa ~2 kd carb 28 kD total Double 52.1 kD aa 26 kD aa  65 Kd 37 kDClip ~4 kD carb ~2 kd carb 56.1 kdD total 28 kd total ¹Estimated MW fromSDS-PAGE relative to Protein MW Markers

Example 27 Generation of Proteolysis-Resistant Variants of ICOSL IgSFDomain-Containing Molecules

To render variant ICOSL polypeptides resistant to proteolysis uponexpression in cells, such as in CHO cells, various additional forms ofvariant ICOSL polypeptides were generated. The following additionalmodified reference sequences of the ICOSL ECD were generated: (1)various ECD truncations lacking all or a portion of the LQQN/LT proteasecleavage site (designated Trunc #4, #5, #6, #7, or #8); (2) ICOSLvariant reference sequences containing mutations at cleavage site N207and/or L208 with reference to positions set forth in SEQ ID NO:32; or anICOSL alone IgV reference sequence containing the IgV domain as the onlyIgSF domain of the molecule (set forth in SEQ ID NO: 545, correspondingto amino acids 1-122 of SEQ ID NO:32). In some cases, combinations ofthe above strategies were employed in a ICOSL ECD reference sequence.Table 30B below sets forth various generated reference sequences.

The exemplary mutations N52H/N57Y/Q100R/F172S, with reference tonumbering set forth in SEQ ID NO: 32, were introduced into the variousreference sequences. Because the reference ICOSL IgV set forth in SEQ IDNO: 545 does not contain a position corresponding to F172S, the variantICOSL IgV did not contain the mutation F172S. The generated variantICOSL polypeptides were formatted as an Fc fusion protein containing thegenerated reference ICOSL IgSF domain linked via a (G₄S)2 linker (SEQ IDNO:229) to an inert Fc containing mutationsC220S/L234A/L235E/G237A/K447del by EU numbering set forth in SEQ ID NO:633, or an allotype thereof set forth in SEQ ID NO:637.

TABLE 30B Exemplary ICOSL IgSF-containing domain reference sequencesReference ICOSL Sequence...---------ECD----|--------Stalk---------------- Full ECD...VNIGCCIENVLLQQNLT VGSQTGNDIGERDKITENPVSTGEKNAAT (32)Truncation #2(600) ...VNIGCCIENVLLQQNL Truncation #3(601)...VNIGCCIENVLLQQNLT VGSQ Truncation #4(602) ...VNIGCCIENVLLQQNTruncation #5(603) ...VNIGCCIENVLLQQ Truncation #6(604) ...VNIGCCIENVLLTruncation #7(605) ...VNIGCCIEN Truncation #8 with ...VNIGCCIENVLLQQ GGT N207G/L208G (623) ECD with N207A (624) ...VNIGCCIENVLLQQ A LTVGSQTGNDIGERDKITENPVSTGEKNAAT ECD with N207G/L208G ...VNIGCCIENVLLQQ GGTV GSQTGNDIGERDKITENPVSTGEKNAAT (628) IgV (545) ...HVAANFSV

A. Proteolysis Assessment

DNA constructs encoding the variant ICOSL Fc-fusion molecules describedabove were transfected into Chinese hamster ovary cells (ExpiCHO-S). TheICOSL Fc-fusion proteins were then purified from supernatants withProtein A by affinity chromatograpy as described in Example 5. Purifiedprotein was analyzed by analytical SEC.

By SEC, intact protein displayed as a single peak while clipped proteindisplayed as multiple peaks, including lower molecular weight species.Consistent with the SDS-PAGE results described in Example 26,proteolysis as assessed by SEC was observed when variant ICOSL ECDFc-fusion protein was expressed in ExpiCHO-S derived cells, as indicatedby the multiple peaks shown in FIG. 22A. As shown in FIGS. 22B-22G,single peaks were observed by SEC analysis of the variant ICOSL Fcfusion proteins generated using modified reference ICOSL polypeptides inwhich the putative ECD protease cleavage site was removed or mutated,indicating reduced cleavage of the proteins occurred. In onepurification lot, however, lower molecular weight species were observedby SEC analysis of the variant ICOSL Fc fusion protein generated usingthe reference ICOSL polypeptide set forth in SEQ ID NO: 604 (Trunc. #5),although it was not clear the reason for the presence of these speciesin this lot. As shown in FIG. 22G, the generation of lower molecularweight species, and hence proteolysis, also was not observed by SECanalysis of the variant ICOSL Fc-fusion proteins generated using theICOSL IgV alone reference sequence.

B. Binding and Activity

Binding and activity of protein produced and purified followingtransfection of DNA constructs encoding the variant ICOSL Fc-fusionimmunomodulatory proteins in various reference sequences described abovein CHO cells was compared. In some cases, purified clones as assessedbelow were later found to contain additional mutations beyond thosedescribed above, which were not believed to impact the immunomodulatoryactivity of the tested proteins.

The resulting purified variant ICOSL Fc-fusion immunomodulatory proteinswere assessed for binding to cognate binding partners and for modulationof T cell activity using a mixed lymphocyte reaction (MLR) substantiallyas described above. Table 30C indicates amino acid substitutions in theICOSL reference sequence as designated by amino acid position numbercorresponding to amino acid positions in the respective reference (e.g.,unmodified) ICOSL extracellular domain (ECD) sequence set forth in SEQID NO: 32, and sets forth the SEQ ID NO identifier for each ICOSLreference sequence. As shown, the binding and MLR antagonist activitywas generally similar for all tested formats.

TABLE 30C Molecule sequences, binding data, and costimulatorybioactivity data of variant ICOSL molecules MLR Binding IFN- ReferenceCD28 CTLA-4 ICOS gamma Sequence MFI MFI MFI pg/mL Description SEQ ID NO(ΔWT) (ΔWT) (ΔWT) (ΔWT) ICOSL ECD Truncation #7 with 605 88329 206566106493 12 N52H/N57Y/Q100R/F172S and F83S (51.1) (27.3) (1.1) (0.02)ICOSL ECD Truncation #6 with 604 91273 239746 90074 14N52H/N57Y/Q100R/F172S (52.8) (31.6) (0.9) (0.02) ICOSL ECD Truncation #4with 602 80555 320229 107957 9 N52H/N57Y/Q100R/F172S and E200G (46.6)(42.3) (1.1) (0.01) ICOSL ECD Truncation #4 with 602 68599 377254 1328802 N52H/N57Y/Q100R/F172S (39.7) (49.8) (1.3) (0.00) ICOSL ECD Truncation#8 with 606 107837 308427 132654 8 N207G/L208G and (62.4) (40.7) (1.3)(0.01) N52H/N57Y/Q100R/F172S ICOSL IgV with N52H/N57Y/Q100R and 54575304 321613 143141 995 H48R/S54P (43.6) (42.4) (1.4) (1.31) ICOSL ECDwith 32 110407 323219 136060 0 N52H/N57Y/Q100R/F172S (63.9) (42.6) (1.4)(0.00) ICOSL ECD Truncation #7 with 605 38876 83695 54596 761N52H/N57Y/Q100R/C198R and (22.5) (11.0) (0.5) (1.00) E90K/E111G ICOSLECD Truncation #6 with 604 84566 236011 91357 7 N52H/N57Y/Q100R/C198R(49.0) (31.1) (0.9) (0.01) ICOSL ECD Truncation #5 with 603 86289 216071110188 9 N52H/N57Y/Q100R/C198R (50.0) (28.5) (1.1) (0.01) ICOSL ECDTruncation #8 with 606 94156 368471 142900 2 N207G/L208G and (54.5)(48.6) (1.4) (0.00) N52H/N57Y/Q100R/C198R and Y151H ICOSL IgV withN52H/N57Y/Q100R 545 84594 204840 117707 0 (49.0) (27.0) (1.2) (0.00)ICOSL ECD with 32 59179 132894 138555 0 N52H/N57Y/Q100R/C198R (34.3)(17.5) (1.4) (0.00) Wildtype ICOSL ECD 32 1727 7579 100466 757 (1.0)(1.0) (1.0) (1.00)

C. Binding and Activity of Proteins Expressed in 293 (Expi293) or CHOCells

The variant ICOSL Fc fusion proteins, generated based on ICOSL referencesequences described above, were assessed for binding and activityfollowing expression in 293 (Expi293) or CHO cells. In addition, a DNAconstruct encoding exemplary IgSF domain ICOSL variantsN52H/N57Y/Q100R/C198R or N52H/Q100R, in exemplary ICOSL referencesequences as set forth in Table 30D, also were linked to an inert Fccontaining mutations C220S/L234A/L235E/G237A by EU numbering set forthin SEQ ID NO: 477, and were produced and purified following transfectionof 293 or CHO cells with the DNA constructs. In addition, an exemplaryvariant immunomodulatory protein was generated as a monomer in whichcells were transfected with a DNA construct encoding the variant in thevariant ICOSL ECD reference sequence but without fusion with an Fcsequence.

The resulting purified variant ICOSL Fc-fusion proteins or variant ICOSLmonomer were assessed for binding to cognate binding partners and formodulation of T cell activity using a mixed lymphocyte reaction (MLR)substantially as described above. Table 30D indicates amino acidsubstitutions in the reference sequence of the variant ICOSL asdesignated by amino acid position number corresponding to amino acidpositions in the respective reference (e.g., unmodified) ICOSLextracellular domain (ECD) sequence set forth in SEQ ID NO:32, and setsforth the SEQ ID NO identifier for each reference ICOSL sequence. Column3 indicates the cell type (ExpiCHO-S or Expi293) used to produce theICOSL protein. As shown in Table 30D, the results indicate substantiallysimilar binding and activity, whether or not the variant ICOSLimmunomodulatory protein was produced in CHO or 293 cells.

TABLE 30D Molecule sequences, binding data, and costimulatorybioactivity data of variant ICOSL ECD Fc-fusion molecules produced usingvarious cells MLR Reference Binding IFN- Sequence CD28 CTLA-4 ICOS gammaSEQ ID CHO or 293 MFI MFI MFI pg/mL Description NO Material (ΔWT) (ΔWT)(ΔWT) (ΔWT) ICOSL ECD with 32 293 13710 (3.9) 8715 (0.7) 16746 (1.4) 20(0.02) N52H/N57Y/Q100R/C198R ICOSL ECD with 32 CHO 12876 (3.6) 8750(0.7) 7700 (0.7) 16 (0.02) N52H/N57Y/Q100R/C198R ICOSCL ECD withN52H/Q100R 32 293 11664 (3.3) 13429 (1.1) 10284 (0.9) 168 (0.19) ICOSCLECD with 32 293 12900 (3.6) 8179 (0.7) 15956 (1.4) 14 (0.02)N52H/N57Y/Q100R/F172S ICOSCL ECD with 32 CHO 14437 (4.1) 8708 (0.7)12610 (1.1) 21 (0.02) N52H/N57Y/Q100R/F172S ICOSCL IgV with 545 29316618 (4.7) 9674 (0.8) 9377 (0.8) 10 (0.01) N52H/N57Y/Q100R ICOSCL IgVwith 545 CHO 17343 (4.9) 9039 (0.7) 8673 (0.7) 14 (0.01) N52H/N57Y/Q100RICOSL ECD Truncation #4 with 602 293 14710 (4.1) 8841 (0.7) 6893 (0.6)21 (0.02) N52H/N57Y/Q100R/F172S ICOSL ECD Truncation #4 with 602 CHO12743 (3.6) 9000 (0.7) 7606 (0.7) 21 (0.02) N52H/N57Y/Q100R/F172S ICOSLECD Truncation #7 with 605 293 12017 (3.4) 9674 (0.8) 7599 (0.7) 15(0.02) N52H/N57Y/Q100R/F172S ICOSL ECD Truncation #7 with 605 CHO 13043(3.7) 9039 (0.7) 8077 (0.7) 7 (0.01) N52H/N57Y/Q100R/F172S ICOSL ECDMonomer with 32 293 20575 (5.8) 19978 (1.6) 11989 (1.0) 133 (0.15)N52H/N57Y/Q100R no Fc ICOSL ECD Dimer with 32 293 18477 (5.2) 22361(1.8) 12913 (1.1) 119 (0.13) N52H/N57Y/Q100R no Fc Wildtype ICOSL ECD 32293 3556 (1.0) 12121 (1.0) 11690 (1.0) 905 (1.00)

Example 28 Generation of NNK Variant Library of ICOSL IgSF DomainVariants and Assessment of Binding and Activity

Additional variant ICOSL IgSF domain-containing molecules were generatedwith mutations at position 52, 57, and 100 with reference to positionsset forth in SEQ ID NO:32. The variants were generated from an NNKlibrary, where K=T or G, such that the encoding codons encode allpotential amino acids, but prevent the encoding of two stop residues TAAand TGA. The NNK library DNA was introduced into yeast substantially asdescribed in Example 2 to generate yeast libraries. The libraries wereused to select yeast expressing affinity modified variants of ICOSLsubstantially as described in Example 3.

The selected variant ICOSL IgSF domain-containing molecules were furtherformatted as an Fc-fusion protein substantially as described in Example4 except containing the generated ICOSL IgSF domain linked via a GSGGGGSlinker (SEQ ID NO:635) to an inert Fc containing mutationsC220S/L234A/L235E/G237A/K447del by EU numbering set forth in SEQ ID NO:633, or an allotype thereof set forth in SEQ ID NO:637.

The variant ICOSL Fc-fusion immunomodulatory proteins were assessed inbinding studies to assess binding for cognate binding partners. Expi293cells transfected with cognate binding partners, human CD28, ICOS andCTLA4, were used as target cells in binding studies as described inExample 6. MFI of binding of 100 nM of variant ICOSL Fc fusionimmunomodulatory proteins for target cells expressing each bindingpartner was determined and compared for binding of the correspondingreference (e.g., unmodified or wildtype) ICOSL IgV-Fc for the sametarget cell. The costimulatory bioactivity of generated ICOSL variantFc-fusion molecules was also assessed in anti-CD3 (10 mM)coimmobilization assays as described in Example 6. Modulation of T cellactivity by the variant ICOSL Fc-fusion immunomodulatory proteins wasalso determined using a mixed lymphocyte reaction (MLR) with 1 nMICOSL-Fc substantially as described in Example 6. IFN-gamma secretionfrom triplicate wells was determined.

Results for binding and functional activity, based on costimulatorybioactivity or activity in an MLR assay, for exemplary variant ICOSLIgV-Fc fusion molecules are shown in Table 31. The Table below indicatesamino acid substitutions in the variant ICOSL as designated by aminoacid position number corresponding to amino acid positions in therespective reference (e.g., unmodified) ICOSL extracellular domain (ECD)sequence set forth in SEQ ID NO:32. Column 2 sets forth the SEQ ID NOidentifier for each variant IgV domain contained in the variant IgV-Fcfusion molecule. As shown, the ICOSL IgSF (e.g. IgV) domain variantsgenerated with the various combinations of specific mutations atpositions 52, 57, and 100, exhibited altered binding for at least one,and in some cases more than one, cognate binding partner. The last twocolumns of the Table also depict the functional activity of the variantFc-fusion molecules to modulate the activity of T cells based on thecalculated levels of IFN-gamma in culture supernatants (pg/mL) generatedeither i) with the indicated variant IgV-Fc fusion soluble moleculecoimmoblized with anti-CD3 or ii) with the indicated variant IgV-Fcfusion molecule in an MLR assay. The Table also depicts the ratio ofIFN-gamma produced by each variant ECD-Fc compared to the correspondingreference (e.g., unmodified or wild-type) ECD-Fc in both functionalassays. Variant Fc-fusion proteins also exhibited altered immunologicalactivity. Costimulatory signaling of some variant molecules wassubstantially greater compared to wild-type ICOSL. Certain variantsexhibited substantial inhibition of IFN-gamma with very low to nodetectable IFN-gamma produced in the cultures in the MLR assay.

TABLE 31 Additional exemplary variant ICOSL polypeptides MLR Costim IFN-SEQ Binding IFN-gamma gamma ID NO CD28 CTLA-4 ICOS pg/mL pg/mL Mutations(IgV) MFI (ΔWT) MFI (ΔWT) MFI (ΔWT) (ΔWT) (ΔWT) N52A/N57F/Q100S 734156589 (7.00) 255078 (0.77) 241891 (1.14) 1119 (0.68) 0 (0.00)N52A/N57H/Q100S 735 159363 (7.10) 321437 (0.97) 304600 (1.44) 2972(1.80) 0 (0.00) N52A/N57Y/Q100A 736 147258 (6.60) 319745 (0.97) 260713(1.23) 2978 (1.81) 0 (0.00) N52D/N57A/Q100A 737 137882 (6.20) 340186(1.03) 248975 (1.17) 477 (0.29) 134 (0.60) N52D/Q100S 738 95731 (4.30)332743 (1.01) 275097 (1.30) 957 (0.58) 110 (0.49) N52G/Q100A 739 98652(4.40) 97118 (0.29) 303229 (1.43) 296 (0.18) 96 (0.43) N52H/Q100A 740145762 (6.50) 361334 (1.09) 213008 (1.01) 784 (0.48) 37 (0.17)N52M/N57H/Q100S 741 114743 (5.10) 463404 (1.40) 265637 (1.25) 1333(0.81) 0 (0.00) N52M/N57W/Q100P 742 168057 (7.50) 342659 (1.04) 322277(1.52) 1865 (1.13) 0 (0.00) N52Q/N57F 743 131301 (5.90) 366714 (1.11)192206 (0.91) 1403 (0.85) 0 (0.00) N52Q/N57S/Q100A 744 91306 (4.10)315021 (0.95) 262735 (1.24) 290 (0.18) 123 (0.55) N52R/ N57L/Q100A 745118803 (5.30) 402961 (1.22) 307965 (1.45) 709 (0.43) 0 (0.00)N52R/N57Y/Q100P 746 133283 (6.00) 502179 (1.52) 251264 (1.19) 7380(4.48) 0 (0.00) N52R/N57Y/Q100S 747 133454 (6.00) 504037 (1.53) 229271(1.08) 5841 (3.54) 0 (0.00) N52S/N57A/Q100A 748 98153 (4.40) 233184(0.71) 181297 (0.86) 442 (0.27) 52 (0.23) N52S/N57H/Q100E 749 116821(5.20) 302383 (0.92) 257518 (1.22) 8412 (5.11) 132 (0.59)N52S/N57L/Q100S 750 108133 (4.80) 197064 (0.60) 268940 (1.27) 3120(1.89) 0 (0.00) N52S/ N57M/Q100S 751 133604 (6.00) 227615 (0.69) 312088(1.47) 349 (0.21) 199 (0.89) N52S/N57Y/Q100S 752 161330 (7.20) 204577(0.62) 223684 (1.06) 7411 (4.50) 0 (0.00) N52S/N57Y/Q100M 753 156869(7.00) 395350 (1.20) 302569 (1.43) 2954 (1.79) 0 (0.00) N52S/N57Y/Q100V754 126281 (5.70) 304795 (0.92) 218925 (1.03) 1304 (0.79) 0 (0.00)N52T/N57H/Q100S 755 143441 (6.40) 377542 (1.14) 258634 (1.22) 6312(3.83) 0 (0.00) N52T/N57H/Q100A 756 112637 (5.00) 350453 (1.06) 220339(1.04) 2874 (1.74) 0 (0.00) N52T/N57Y/Q100A 757 161333 (7.20) 340845(1.03) 239136 (1.13) 442 (0.27) 0 (0.00) N52V/N57L/Q100A 758 132144(5.90) 252148 (0.76) 181344 (0.86) 518 (0.31) 159 (0.71) N52H/N57Y/Q100K759 141720 (6.30) 393476 (1.19) 214270 (1.01) 12919 (7.84) 0 (0.00)N52K/N57Y/Q100R 760 140729 (6.30) 233283 (0.71) 198941 (0.94) 12515(7.60) 0 (0.00) N52L/N57H/Q100R 761 140807 (6.30) 352518 (1.07) 250052(1.18) 12544 (7.61) 0 (0.00) N52R/N57F/Q100N 762 161029 (7.20) 233254(0.71) 252904 (1.19) 448 (0.27) 106 (0.47) N52R/N57F/Q100P 763 153850(6.90) 503696 (1.52) 296566 (1.40) 1718 (1.04) 0 (0.00) N52R/N57F/Q100R764 185231 (8.30) 463873 (1.40) 234248 (1.11) 11402 (6.92) 0 (0.00)N52R/N57F/Q100T 765 126875 (5.70) 357505 (1.08) 270134 (1.27) 272 (0.17)0 (0.00) N52R/N57H/Q100K 766 — — — — — N52R/N57L/Q100S 767 111704 (5.00)289326 (0.88) 230617 (1.09) 1292 (0.78) 0 (0.00) N52R/N57W/Q100K 768130875 (5.90) 477268 (1.44) 349316 (1.65) 10056 (6.10) 0 (0.00)N52R/N57W 769 136967 (6.10) 318199 (0.96) 298850 (1.41) 12652 (7.68) 0(0.00) N52R/N57Y/Q100R 770 3285 (0.10) 4266 (0.01) 6104 (0.03) 349(0.21) 347 (1.54) N52C/N57E/Q100S 771 13361 (0.60) 10616 (0.03) 125274(0.59) 296 (0.18) 372 (1.65) N52G/N57P/Q100D 772 5715 (0.30) 10181(0.03) 274629 (1.30) 254 (0.15) 343 (1.53) N52G/N57V/Q100G 773 23658(1.10) 14727 (0.04) 260057 (1.23) 325 (0.20) 245 (1.09) N52G/N57V 77469117 (3.10) 52498 (0.16) 332068 (1.57) 847 (0.51) 327 (1.45) N52L/N57V775 54775 (2.50) 150970 (0.46) 256730 (1.21) 986 (0.60) 270 (1.20)N52P/N57P 776 21008 (0.90) 27043 (0.08) 222171 (1.05) 260 (0.16) 478(2.13) N52P/N57S/Q100G 777 6803 (0.30) 5054 (0.02) 143255 (0.68) 110(0.07) 481 (2.14) N52S/N57L/Q100G 778 71895 (3.20) 79432 (0.24) 275602(1.30) 726 (0.44) 513 (2.28) N52T/N57K/Q100P 779 88653 (4.00) 78299(0.24) 312905 (1.48) 116 (0.07) 395 (1.76) N52V/N57T/Q100L 780 6205(0.30) 11458 (0.03) 29167 (0.14) 85 (0.05) 562 (2.50) N57Q/Q100P 78115195 (0.70) 69058 (0.21) 204533 (0.97) 159 (0.10) 432 (1.92) WT ICOSL545 22340 (1.00) 330437 (1.00) 211945 (1.00) 1648 (1.00) 225 (1.00) Fulllength 291 138141 (6.20) 605794 (1.83) 237653 (1.12) — 0 (0.00)N52H/N57Y/Q100R/F172S N52H/N57Y/Q100R 565 142274 (6.40) 817010 (2.47)199528 (0.94) — 0 (0.00) N52H/N57Y/Q100R 283 — — — 10362 (6.29) —

Example 29 Assessment of K562 Cells Expressing a TransmembraneImmunomodulatory Protein (TIP)

K562 cells were engineered to express a Transmembrane ImmunomodulatoryProtein (TIP) that contained the ECD of the vIgD ICOSL domains fused tothe native transmembrane and intracellular domain of WT human ICOSL setforth in SEQ ID NO: 5. The exemplary variant ICOSL-TIP had anaffinity-modified IgSF domain containing amino acid mutationscorresponding to N52H/N57Y/Q100P (SEQ ID NO: 288), N52H/N57Y/Q100R (SEQID NO: 283), or E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R (SEQID NO: 300) with reference to positions in the ICOSL extracellulardomain set forth in SEQ ID NO:32.

K562 cells (ATCC) were labeled with CFSE to better distinguish them fromT cells in co-culture assays. Purified primary human T cells werelabeled with Cell Trace Far Red (both from Thermo-Fisher) and co-platedin a 96 well round bottom tissue culture plates with anti-CD3 antibody.To provide a TCR signal for T cells, anti-CD3 antibody was included insoluble format over a range of concentrations that allowed K562presentation of this stimulating antibody through the Fc-receptor CD32expressed by the cells. Cells were incubated 72 hours and proliferationof CD4+(FIG. 23A) and CD8+(FIG. 23B) was monitored and reported aspercent of cells divided versus anti-CD3 concentration. Each pointrepresents the mean of triplicate weels with error bars showing standarddeviation.

Wild type K562 cells stimulated T cells to proliferate when co-incubatedwith soluble anti-CD3 antibody in a dose-dependent manner, whereas K562cells in the absence of anti-CD3 did not. As shown in FIGS. 23A and 23B,expression of WT ICOSL TIP on the surface enhanced responses, but theeffects were greater when K562 cells expressed the variant ICOSL TIPs,indicating that these molecules expressed on the surface of cellsprovided superior costimulation for T cells.

Example 30 Assessment of Binding and Costimulatory Function of FusionMolecules with HER2-Targeting Antibody

An exemplary variant ICOSL ECD, containing mutations N52H/N57Y/Q100R wasfused to either the N- or C-terminal ends of the heavy or light chain ofthe exemplary anti-HER2 antibody, trastuzumab, as depicted in thevarious configurations shown in FIG. 24A-24F. VmAbs DNA encoding each ofthe constructs diagrammed in FIG. 24A-24F was transfected into HEK-293cells and secreted proteins were purified by Protein A and sizeexclusion chromatography. The resultant V-mAb proteins were nextassessed for retention of appropriate binding properties. HEK-293 cellswere transiently transfected with HER2, CD28, or ICOS expression vectorsand each transfectant was then incubated with individual V-mAb proteinsplus a secondary antibody for detection of bound reagents. As shown inFIG. 24A, HER2 binding was retained by all V-mAbs although the magnitudeof the binding was reduced somewhat. Moreover, binding of V-mAbs to CD28transfected cells was largely intact, although a few forms showed somereduction in binding (FIG. 24A). This data indicated that the ICOSLvariants fused to antibody heavy and/or light chains to form fusionproteins largely retained counter-structure and antibody bindingactivity.

To test whether VmAbs could drive target-specific costimulation of Tcells, a transfected cell system including a T cell reporter line formeasuring costimulation was used. Jurkat cells with an IL-2 promoterluciferase reporter were used to evaluate costimulatory function. Tostimulate the cells, K562 cells were engineered for use as an artificialantigen presenting cell. Specifically, K562 cells were transduced with alentivirus encoding a single-chain Fv version of the anti-CD3 antibodyOKT3 with or without transduction with a separate lentivirus directingHER2 expression. K562 cells displaying cell surface anti-CD3 singlechain Fv (OKT3) with or without surface HER2 expression were plated inJurkat Assay Buffer (RPMI1640+5% FBS) at 2×10⁴ cell/well. Target cellswere incubated with V-mAbs titrated from 20,000 pM to 6 pM or controlproteins for 20 minutes at room temperature. Jurkat effector cellsexpressing an IL-2-luciferase reporter gene (Promega) were added at1×10⁵ cell/well to bring the final volume/well to 100 μl. Target andJurkat cells were incubated for 5 hours at 37° C. Plates were removedfrom the incubator and acclimated to room temperature for 15 minutes.100 μL of cell lysis and luciferase substrate solution (BioGloluciferase reagent, Promega) was added to each well and the plates wereincubated on an orbital shaker for 10 minutes. Luminescence was measuredwith a 1 second per well integration time using a Cytation 3 imagingreader (BioTek Instruments). Relative luminescence values (RLU) weredetermined for each test sample and reported.

As shown in FIG. 24B, inclusion of native trastuzumab had no effect onluciferase induction. Similarly, inclusion of the variant ICOSL-Fcprotein N52H/N57Y/Q100R (not fused to trastuzumab) did not affectresponses (FIG. 24B). However, inclusion of multiple V-mAbs provided asignificant costimulatory signal in the presence of HER2+K562/OKT3 cellsthat was much more robust than with K562/OKT3 that lacked HER2expression (FIG. 24C-24F). In some cases, signal was induced inK562/OKT3 cells lacking HER2, but this was most likely due to the Fcdomain of the V-mAbs allowing CD32-mediated presentation of the V-mAbs.The results indicate that fusing a variant ICOSL polypeptide to anantibody can be used to deliver a localized T cell costimulatory signal.

Example 31 Generation of Stacked Molecules Containing ICOSL and NKp30Affinity-Modified Domains

This Example describes immunomodulatory proteins that were generated asmulti-domain stack constructs containing an affinity modified IgV domainfrom identified variant ICOSL polypeptides and identified variant NKp30polypeptides described above. Specifically, an exemplary variant ICOSLIgV (N52D as set forth in SEQ ID NO:548; N52H/Q100R as set forth in SEQID NO:567; N52H/N57Y/Q100R as set forth in SEQ ID NO: 565;N52L/N57H/Q100R as set forth in SEQ ID NO: 761), and the exemplaryvariant NKp30 IgV molecule L30V/A60V/S64P/S86G (SEQ ID NO: 504) werelinked together and fused to an inert Fc (containing mutations L234A,L235E and L235E in a human IgG1 Fc, e.g. set forth in SEQ ID NO:637) invarious configurations. Homodimeric stack constructs were generatedcontaining identical Fc subunits in which the variant ICOSL IgV andvariant NKp30 IgV were variously linked to the N- or C-terminus of theFc region via a GSGGGS (SEQ ID NO: 635) and/or 3×GGGGS (SEQ ID NO: 228)peptide linker. Other linkers and Fc regions also are suitable forgeneration of stack molecules. Exemplary generated stacks are set forthbelow.

Nucleic acid molecule encoding the immunomodulatory proteins alsocontained residues encoding the exemplary signal peptideMGSTAILALLLAVLQGVSA (set forth in SEQ ID NO: 225). Expression constructsencoding Fc fusion proteins of interest were transiently expressed inExpi293 HEK293 cells from Invitrogen using the manufacturer's commercialExpifectamine reagents and media. Supernatants were harvested andprotein was captured and eluted from a Protein A column using an AKTAprotein purification system.

The encoding nucleic acid molecule was designed to produce homodimericstacks in various configurations of sequences in the order shown inTable 32.

TABLE 32 Description of ICOSL/NKp3Q Immunomodulatory Proteins ProteinSEQ ID NO Stack (DNA SEQ ID NO) Description ICOSL/ 912 ICOSL variant(SEQ ID NO: 548) - NKp30 (911) 3x GGGGS (SEQ ID NO: 228) - Stack 1 NKp30variant (SEQ ID NO: 504 ) - GSGGGGS (SEQ ID NO: 635) - Fc (SEQ ID NO:637) ICOSL/ 914 ICOSL variant (SEQ ID NO: 548) - NKp30 (913) 3x GGGGS(SEQ ID NO: 228) - Stack 2 NKp30 variant (SEQ ID NO: 504) - 3x GGGGS(SEQ ID NO: 228) - NKp30 variant (SEQ ID NO: 504) - GSGGGGS (SEQ ID NO:635) - Fc (SEQ ID NO: 637) ICOSL/ 916 ICOSL variant (SEQ ID NO: 567) -NKp30 (915) 3x GGGGS (SEQ ID NO: 228) - Stack 3 NKp30 variant (SEQ IDNO: 504) - GSGGGGS (SEQ ID NO: 635) - Fc (SEQ ID NO: 637) ICOSL/ 918ICOSL variant (SEQ ID NO: 567) - NKp30 (917) 3x GGGGS (SEQ ID NO: 228) -Stack 4 NKp30 variant (SEQ ID NO: 504) - 3x GGGGS (SEQ ID NO: 228) -NKp30 variant (SEQ ID NO: 504) - GSGGGGS (SEQ ID NO: 635) - Fc (SEQ IDNO: 637) ICOSL/ 920 ICOSL variant (SEQ ID NO: 565) - NKp30 (919) 3xGGGGS (SEQ ID NO: 228) - Stack 5 NKp30 variant (SEQ ID NO: 504) -GSGGGGS (SEQ ID NO: 635) - Fc (SEQ ID NO: 637) ICOSL/ 922 ICOSL variant(SEQ ID NO: 565) - NKp30 (921) 3x GGGGS (SEQ ID NO: 228) - Stack 6 NKp30variant (SEQ ID NO: 504) - 3x GGGGS (SEQ ID NO: 228) - NKp30 variant(SEQ ID NO: 504) - GSGGGGS (SEQ ID NO: 635) - Fc (SEQ ID NO: 637) ICOSL/924 ICOSL variant (SEQ ID NO: 761) - NKp30 (923) 3x GGGGS (SEQ ID NO:228) - Stack 7 NKp30 variant (SEQ ID NO: 504) - GSGGGGS (SEQ ID NO:635) - Fc (SEQ ID NO: 637) ICOSL/ 926 ICOSL variant (SEQ ID NO: 761) -NKp30 (925) 3x GGGGS (SEQ ID NO: 228) - Stack 8 NKp30 variant (SEQ IDNO: 504) - 3x GGGGS (SEQ ID NO: 228) - NKp30 variant (SEQ ID NO: 504 ) -GSGGGGS (SEQ ID NO: 635) - Fc (SEQ ID NO: 637)

Example 32 Assessment of Binding to Cell-Expressed Counter Structuresand Bioactivity of ICOSL and NKp30 Domain-Containing Stack Molecules

This Example describes binding studies to show specificity and affinityof exemplary ICOSL/NKp30 stack immunomodulatory proteins generated inExample 31 for cognate binding partners. The exemplary ICOSL/NKp30 stackimmunomodulatory proteins generated in Example 31 also were assessed forbioactivity characterization in a human primary T cell in vitro assay.

A. Binding to Cell-Expressed Counter Structure

ICOSL/NKp30 stack binding studies were performed on cells with stable ortransient cell surface expression of cognate binding partners specificfor ICOSL domain variant immunomodulatory proteins or NKp30-Fc.

For assessing binding to a binding partner of variant ICOSL domains,Chinese hamster ovarian cells (CHO) were used which had been transducedwith lentivirus for surface expression of full-length human CD28,CTLA-4, or ICOS.

To produce cells expressing the cognate binding partner of NKp30, afull-length mammalian surface expression construct containing humanB7-H6 was cloned into a pcDNA3.1 expression vector (Life Technologies).Binding studies were carried out using the Expi293F transienttransfection system (Life Technologies, USA). Briefly, for a 30 mLtransfection, approximately 75 million Expi293F cells were incubatedwith 30 μg of expression construct DNA and 1.5 mL diluted ExpiFectamine293 reagent for 48 hours, at which point cells were harvested forstaining.

For flow cytometric analysis, 200,000 cells of a given stable cell line,transient transfection or appropriate negative control were plated in 96well round bottom plates. Cells were spun down and suspended in stainingbuffer (PBS (phosphate buffered saline), 1% BSA (bovine serum albumin),and 0.1% sodium azide) for 20 minutes to block non-specific binding.Afterwards, cells were centrifuged again and suspended in stainingbuffer containing 100 nM to 32 pM of ICOSL/NKp30 stack or controlprotein in 50 μL. Primary staining was performed for 45 minutes, beforewashing cells in staining buffer twice. Bound protein was detected withPE-conjugated anti-human IgG (Jackson ImmunoResearch, USA) diluted 1:150in 50 μL staining buffer and incubated for 30 minutes. After finalincubation, cells were washed twice to remove unbound conjugatedantibodies, fixed in 2% formaldehyde/PBS, and analyzed on a LSRII(Becton Dickinson, USA) flow cytometer. Mean Fluorescence Intensity(MFI) was calculated for each sample with FlowJo Version 10 software(FlowJo LLC, USA).

Binding activity as measured by MFI was assessed for all ICOSL-NKp30 Fcfusion proteins or controls. As shown in FIG. 25A-25D, exemplary stackproteins bound cognate proteins of both ICOSL and NKp30 with highaffinity.

B. Assessment of Bioactivity of Affinity-Matured IgSF Domain-ContainingMolecules

Soluble ICOSL/NKp30 stack protein bioactivity was tested in a co-culturewith B7-H6+ cells for induction of cytokine production in primary humanT cells. K562 cells which endogenously express B7-H6 were transducedwith lentivirus to express cell surface anti-human CD3 single chain Fv(OKT3) yielding K562/OTK3 targets. Human primary T cells wereco-cultured at an effector to target (E:T) ratio of 2.5 or 10:1 withICOSL/Nkp30 stack or control proteins titrated from 100 nM to 49 pM in200 μL final volume of Ex-Vivo 15 media. On day 3-5, the assay wasterminated and the culture supernatants were tested using IL-2 andTNF-alpha ELISA MAX kits (Biolegend, USA). Optical density was measuredon a BioTek Cytation Multimode Microplate Reader (BioTek Corp., USA) andquantitated against titrated rIL-2 and rTNF-alpha standards included inthe ELISA kits.

Results for the bioactivity studies for exemplary tested ICOSL/NKp30stack proteins are shown in FIGS. 26A and 26B, which sets forth thecalculated levels of IL-2 or IFN-gamma in culture supernatants (pg/mL).The sequence identifier (SEQ ID NO) for each stack proteins is set forthin FIGS. 26A and 26B. Incubation in the presence of exemplaryICOSL/NKp30 stack proteins in this assay resulted in increased levels ofB7-H6-dependent cytokine induction in primary human T cells demonstratedby an increase in cytokine production with the ICOSL/NKp30 stackscompared to the parental ICOSL or NKp30 only proteins.

C. Assessment of Proliferation

Proliferation of human T-cells co-cultured with ICOSL/NKp30 stackproteins and B7-H6+ cells was also characterized. CFSE-labeled humanprimary T-cells were stimulated for 3-5 days with K562/OKT3 at an E:Tratio of 2.5 to 10:1 in the presence of ICOSL/NKp30 stack proteins orcontrol proteins. Exemplary ICOSL/NKp30 stack proteins were titratedfrom 100 nM to 49 pM in 200 μL final volume of Ex-Vivo 15 media.Proliferation was measured by flow cytometric analysis of CFSE-dilutionon CD4+ or CD8+ stained T-cells using LSRII flow cytometer and Flowjosoftware as described above.

As shown in FIG. 27, exemplary tested ICOSL/NKp30 stack proteinscostimulated proliferation of primary human CD4+ T cells in aB7H6-dependent manner demonstrated by an increase in proliferation ofthe ICOSL/NKp30 stack compared to the parental ICOSL or NKp30 onlyproteins.

Example 33 Assessment of ICOSL and NKp30 Domain-Containing StackMolecules in Combination and Anti-PD-1 Antibody in Tumor Model

This Example describes the assessment of anti-tumor activity of anexemplary ICOSL/NKp30 stack proteins, generated as described in Example31, evaluated alone or in combination with an anti-mouse PD-1 monoclonalantibody (mPD-1 mAb) in mice bearing B7-H6+CT26 colon carcinoma cells.

Mice were implanted subcutaneously with approximately 0.3×10⁵ B7-H6+CT26tumor cells. Tumors were grown to day 13 and mice were staged andmeasured for mean tumor volumes (80 to 120 mm³). Tumors were measuredwith electronic calipers two-dimensionally beginning on day 6 post-tumorcell implant. Tumor volume was measured, and median tumor volume wasdetermined. Three mice/group with smallest starting tumors (˜75 mm³)were excluded from the analysis.

As shown in FIG. 28, the combination of the tested ICOSL/NKp30 stackprotein and mPD-1 mAb significantly reduced tumor growth (median tumorvolumes) over time compared to groups treated with Fc control, eitherICOSL or NKp30 alone, ICOSL/NKp30 stack alone, or anti-mouse PD-1 mAbalone. No differences in outgrowth of any treatment group with parentalCT26 (B7-H6-negative) tumors was observed. The anti-tumor activity ofthe combination as shown in FIG. 28 is consistent with a finding thatthe combination of the tested ICOSL/NKp30 stack protein and anti-PD-1antibody is better than individual reagents alone.

Example 34 Assessment of Dosing and In Vivo Effects of ICOSL IgV-FcFusion Molecules in a CIA Model

Variant ICOSL IgV-Fc fusion molecules were assessed foranti-inflammatory activity in the collagen-induced arthritis (CIA) modelwith either prophylactic or therapeutic dosing. The variant ICOSL IgV-Fcfusion molecule was dosed a maximum of 4 times either prior to or justafter disease onset. The tested variant ICOSL IgV-Fc fusion moleculecontained a variant ICOSL IgV with N52H/N57Y/Q100P as set forth in SEQID NO: 570 or N52H/N57Y/Q100R as set forth in SEQ ID NO: 565, fused toan inert Fc (containing mutations L234A, L235E and L235E in a human IgG1Fc, e.g. set forth in SEQ ID NO:637).

For induction of joint inflammation, mice were injected on day −18 or−21 with a chick or bovine collagen II/CFA emulsion in the tail and witha chick or bovine collagen II/IFA emulsion (‘boost’) on day 0. Forprophylactic dosing, mice were dosed with the variant ICOSL IgV-Fcfusion molecule (N52H/N57Y/Q100P) with four doses beginning on the dayof the boost, before the onset of disease. For therapeutic/delayedtreatment, mice were dosed with the variant ICOSL IgV-Fc fusion molecule(N52H/N57Y/Q100R) started when the observed paw score was greater thanone, and dosing occurred every two days for a total of four doses. As acontrol, Fc only molecules and a CTLA-4-Fc (abatacept) molecule werealso tested. Paw score based on redness or swelling was determined.Serum also was collected to measure anti-collagen (CII IgG) antibodiesand IL-6 and TNFα proinflammatory cytokines. Cells from draining lymphnodes were collected, stained for CD4, CD8, CD44 or markers of Tfollicular helper (T_(FH)) cells (CD25-CD4+PD-1+CXCR5+). FIG. 29A-29Dshow results for prophylactic dosing. Mice treated with the variantICOSL IgV-Fc fusion molecule on the prophylactic dosing treatment showedsuppressed disease in the CIA mouse model of rheumatoid arthritis asshown by a lower mean sum paw score (FIG. 29A), and decreased detectedCII IgG (FIG. 29B). *p<0.05 for ICOSL IgV-Fc vs. abatacept **p<0.001 forICOSL IgV-Fc vs. PBS (by 2-way repeated-measures ANOVA). Lower levels ofserum cytokines (FIG. 29C) and CD44+ activated T cells or T_(FH) cells(FIG. 29D) were also observed in mice treated with the variant ICOSLIgV-Fc fusion molecule compared to Fc control; *p<0.05, **p<0.01,***p<0.001, ****p<0.0001 (by 1-way ANOVA). The fraction of B cells inthe draining lymph node was also significantly reduced in the ICOSLIgV-Fc treated group vs. the Fc control group (p<0.05) (FIG. 29E).

FIG. 30A-30D show results for delayed dosing. The variant ICOSL-IgV Fcresulted in the lowest mean sum paw score (FIG. 30A) and greatestpercent change in body weight (FIG. 30B) compared to other groups,including the abatacept control. As shown in FIG. 30C and FIG. 30D,serum cytokines also were suppressed in the therapeutic CIA model inmice treated with the variant ICOS IgV-Fc. Statistical significancebetween groups: *p<0.05; **p<0.01; ***p<0.001 by Student's unpairedt-test. In FIG. 30C and FIG. 30D, the dotted horizontal lines indicatethe assay lower limit of quantification (LLOQ) for each cytokine.

Together, these data evidence that the CD28 and ICOS pathways playimportant roles in inflammatory arthritis. In particular, the superioractivity of the variant ICOSL dual CD28/ICOS antagonist is consistentwith an observation that blockade of both pathways is necessary and thatonly partial benefit is achieved by a single pathway blockade.

Example 35 Assessment of In Vivo Effects of ICOSL IgV-Fc FusionMolecules in a EAE Model

A variant ICOSL IgV-Fc fusion molecule was assessed foranti-inflammatory activity in an adoptive transfer experimentalautoimmune encephalomyelitis (EAE) model. The tested variant ICOSLIgV-Fc fusion molecule containing a variant ICOSL IgV (N52H/N57Y/Q100R;SEQ ID NO: 565) fused to an inert Fc (containing mutations L234A, L235Eand L235E in a human IgG1 Fc).

Female C57BL/6 mice were subcutaneously injected with a MOG₃₅₋₅₅/CFAemulsion. After 11 days, spleen cells were obtained and cultured withMOG₃₅₋₅₅ peptide, IL-12, and anti-IFNγ. Three days post culture,encephalitogenic T cells were delivered via intraperitoneal injection(Day 0). Mice were dosed with variant ICOSL IgV-Fc fusion molecule everyother day starting on Day 0 for a total of five doses. As a control, Fconly molecules and a CTLA-4-Fc molecule (abatacept) were also tested.For 20 days post injection of the T cells, mice were weighed, monitoredand assessed for EAE score as described in Table 33. At end of study,serum was collected for analysis of pro-inflammatory cytokines, andcells from draining lymph nodes were collected for flow cytometricanalysis.

TABLE 33 EAE Scoring Score Clinical Observations 0 No obvious changes inmotor functions 1 Limp tail 2 Limp tail and weakness of hind legs 3 Limptail and complete paralysis of hind legs, OR Limp tail with paralysis ofone front and one hind leg, OR ALL of: 1) Severe head tilting, 2)Walking only along the edges of the cage, 3) Pushing against the cagewall, 4) Spinning when picked up by the tail. 4 Limp tail, complete hindleg and partial front leg paralysis 5 Complete hind and complete frontleg paralysis, no movement; OR Mouse is spontaneously rolling in thecage; OR Mouse found dead due to paralysis.

As shown in FIG. 31A, mice treated with the variant ICOSL IgV-Fc fusionmolecule suppressed disease in the EAE mouse model as shown by a lowerEAE score, *p<0.0001 by 1-way ANOVA Area Under the Curve (AUC); variantICOSL IgV-Fc fusion molecule compared to controls.

For flow cytometric analysis of inguinal lymph node T cells, the cellswere stained with viability dye & analyzed with anti-CD44, anti-CD62L,anti-CD4, anti-CD8 and assessed for percentage of viable naïve(CD62L+CD44-) and T_(effector) memory (Tem) (CD62L-CD44+) CD4+ and CD8+T cells. As shown in FIG. 31C, CD4+ and CD8+ Tem cells were reduced withtreatment with the variant ICOSL IgV-Fc fusion molecule (****p<0.0001;***p<0.001 by 1-way ANOVA).

Serum cytokines were assessed on Day 0 (2 hours post 1^(st) dose) and onDay 6 (1 hour before 4^(th) dose). As shown in FIG. 31D, the testedvariant ICOSL IgV-Fc fusion molecule resulted in reduction ofpro-inflammatory cytokines in serum on Day 0, including IL-5, IL-10,IL-12p70 and TNFα. At day 6, serum levels of IFN-gamma and IL-6 werereduced by treatment with the variant ICOSL IgV-Fc fusion moleculecompared to Fc control.

Example 36 Dose Ranging Study of Variant ICOSL-IgV Fc inGraft-Versus-Host-Disease (GvHD) Model

A dose ranging study was conducted with 20, 100, or 500 μg of a variantICOSL IgV-Fc molecule, containing a variant ICOSL IgV (N52H/N57Y/Q100R;SEQ ID NO: 565) fused to an inert Fc (containing mutations L234A, L235Eand L235E in a human IgG1 Fc, e.g. set forth in SEQ ID NO:637), in amouse model of graft-versus-host-disease (GVHD). The activity of thevariant ICOSL IgV-Fc molecule was compared to belatacept(CTLA-4-FcL104E/A29Y; U.S. Patent Application Publication NumberUS2016/0271218).

Female NSG mice (n=5 per group for Group 1, no treatment; n=10 per groupfor treatment Groups 2-7) were administered 10 mg of gamma globulinsubcutaneously and then irradiated (100 cGy/rad) on Day −1. On Day 0(within 24 hours post-irradiation), the mice in Groups 2-7 were dosedwith test articles as set forth in Table 34, and then all mice received1×10⁷ human PBMCs injected IV via tail vein post-dosing.

A disease activity index (DAI) was determined by evaluating the micethree times a week during the study and scoring disease based on bodyweight loss, posture, activity, appearance of the fur and skin of themice. After the study was terminated on Day 42, endpoint measurements ofsurvival, body weight loss, and disease activity were evaluated.Kaplan-Meier survival plots representing the percentage of animalssurviving to the study endpoint were generated and survival curvecomparisons were analyzed by the Mantel-Cox and Gehan-Breslow-Wilcoxtests (95% CI). Blood/serum samples were collected from surviving miceat the end of the study (Day 42) and cells were assessed by flowcytometry for markers of T cells, including mouse or human markers, CD4,CD8, CD28, ICOS, activation or exhaustion markers (PD-1, Ki67) and FoxP3(a marker of Tregs). The levels of serum pro-inflammatory cytokines(e.g. IFN-gamma, IL-10, IL-12(p70), IL-17A, IL-4, IL-5 and TNFα) alsowere assessed.

TABLE 34 Dosing Schedule Dose Dose Volume Start of Group N Test Articles(μg) (μl) Route Schedule Dosing 1 5 No treatment n/a n/a n/a n/a n/aSurvival/DAI 2 10 Saline n/a 100 IP 3x weekly Day 0 4 weeks 3 10 variantICOSL IgV-Fc 500 100 IP 3x weekly Day 0 4 weeks 4 10 variant ICOSLIgV-Fc 100 100 IP 3x weekly Day 0 4 weeks 5 10 variant ICOSL IgV-Fc 20100 IP 3x weekly Day 0 4 weeks 6 10 Belatacept 100 100 IP 3x weekly Day0 4 weeks PK/Survival//DAI 7 9 variant ICOSL IgV-Fc 100 100 IP SingleDay 1 Day 1

FIG. 32A-32B show the survival and DAI scores of GVHD mice treated inaccord with each dosing schedule. As shown, the tested variant ICOSLIgV-Fc (N52H/N57Y/Q100R) at all dose levels tested significantlyenhanced survival (FIG. 32A) and reduced disease scores (FIG. 32B)compared to mice treated with belatacept (i.e. 100% vs. 40% survival atDay 42, respectively; p<0.01 by Mantel-Cox log rank test). Notably,single dose (100 μg) administration of variant ICOSL IgV-Fc(N52H/N57Y/Q100R) resulted in similar protection from disease as repeatdosing of 100 μg belatacept.

Flow cytometric analysis of blood collected at the end of the studydemonstrated that the tested variant ICOSL IgV-Fc (N52H/N57Y/Q100R)effectively suppressed expansion of transferred human T cells asobserved by a reduced ratio of human cells/mouse cells (FIG. 33A) andthe greatly reduced total T cell counts (FIG. 33B). As shown in FIGS.33C-33F, flow cytometry staining of blood collected at end of the studyof CD4+ and CD8+ T cells co-stained for ICOS (FIGS. 33C-33D) and CD28(FIGS. 33E-33F) demonstrated essentially no staining in groups treatedwith variant ICOSL IgV-Fc (N52H/N57Y/Q100R), although CD4+ and CD8+ Tcells expressing ICOS were readily detectable in belatacept-treatedmice. These results are consistent with the lack of T cells remaining inthe ICOSL IgV-Fc-treated mice, and also with the ability of the variantICOSL IgV-Fc (N52H/N57Y/Q100R) to bind its target molecules CD28 andICOS and block their detection by the flow cytometry antibodies. Bindingof variant ICOSL IgV-Fc (N52H/N57Y/Q100R) on the few remaining human Tcells was confirmed by detection using anti-Human IgG Fc. Notably, whilemost of the transferred human T cells initially expressed CD28 and just10-20% were ICOS+, the activated T cells remaining in the saline- orbelatacept-treated mice at termination/end of study were >80% ICOS+.

The presence of activation or exhaustion markers of T cells also wassuppressed in groups treated with variant ICOSL IgV-Fc(N52H/N57Y/Q100R), as evidenced by lower expression of PD1 in CD4+ andCD8+ T cells and decreased Ki67 expression in CD4+ T cells, (FIG.34A-3B). The ratio of effector T cells to Tregs remained stable ingroups treated with variant ICOSL IgV-Fc (N52H/N57Y/Q100R) compared tobelatacept (FIG. 34C). Serum proinflammatory cytokines also weresuppressed in groups treated with variant ICOSL IgV-Fc (N52H/N57Y/Q100R)compared to belatacept (FIG. 35A-35D).

Pharmacokinetic (PK) analysis also was carried out to monitor serumexposure of variant ICOSL IgV-Fc (N52H/N57Y/Q100R). Test articleconcentrations were measured in mouse serum samples using a quantitativePK ELISA using an anti-human ICOSL mAb capture antibody and anFc-specific mouse anti-human IgG as the detection reagent.

The observed serum exposure of variant ICOSL IgV-Fc (N52H/N57Y/Q100R) inthe GVHD model was 45% lower than that of normal mice, determined in aseparate study (FIG. 35E). The longer terminal half-life of variantICOSL IgV-Fc (N52H/N57Y/Q100R) in the GvHD model may due to reducedtarget (CD28, ICOS) at later time points in GvHD (as the human T cellsdisappear), and/or to anti-drug antibody (ADA) formation in normal mice,which can interfere with drug exposure. The observation that variantICOSL IgV-Fc (N52H/N57Y/Q100R) had lower serum exposure compared tonormal mice may be due to target mediated drug disposition (TMDD) in theGvHD model (i.e. its higher affinity for human CD28 and ICOS as comparedto the mouse orthologues), and/or to lack of FcRn in the NOD/SCID (NSG)mice used in this model.

Together, these results are consistent with an observation that variantICOSL IgV-Fc (N52H/N57Y/Q100R) exhibits potent antagonist activity, evenwith only a single dose, and superior activity to belatacept. Thisobservation may be attributable to the variant ICOSL IgV-Fc(N52H/N57Y/Q100R) exhibiting superior control of ICOS+ T cells, whichotherwise escape single ICOS or CD28 pathway blockade, such as thatachieved with the CD28 pathway antagonist belatacept.

Example 37 Assessment of Variant ICOSL-IgV Fc in CD4+CD45RBhigh-InducedColitis Model

The effect of the exemplary variant ICOSL IgV-Fc containing a variantICOSL IgV (N52H/N57Y/Q100R; SEQ ID NO: 565) fused to an inert Fc(containing mutations L234A, L235E and L235E in a human IgG1 Fc, e.g.set forth in SEQ ID NO:637) on disease development inCD4+CD45RBhigh-induced colitis model was evaluated.

CD4+CD45RBhigh donor cells were enriched by negative selection fromspleen cell suspensions obtained from 15 male BALB/C donor mice. On Day0, 0.3 million CD4+CD25-CD45RBhigh (Treg depleted) donor cells wereinjected intravenously into immunodeficient C.B17 (SCID) mice (n=12 or21 per group) to induce colitis. As a control, 0.3 million CD4+ cells(containing Treg cells), which do not induce development of colitis inthis model, were injected into SCID mice recipients (n=12). On the dayof cell transfer, mice in each group were dosed with variant ICOSL-IgVFc or Fc only or vehicle controls. Table 35 summarizes the treatmentregimen for tested groups.

TABLE 35 Treatment Regimens # Group mice Cells injected Treatment DoseRoute Frequency 1 12 CD4+ PBS (sterile) — i.p. 3x/week (M, W, F) 2 12CD4 + CD45RBhigh PBS (sterile) — i.p. 3x/week (M, W, F) 3 21 CD4 +CD45RBhigh Fc control 300 μg i.p. 3x/week (M, W, F) 5 12 CD4 +CD45RBhigh Variant ICOSL 400 μg i.p. 3x/week IgV-Fc (M, W, F)

Body weight (taken starting on Day 0) and stool consistency score (takenstarting on Day 10), were evaluated three times per week. Daily diseaseactivity index (DAI) was calculated from body weight and stool scores.After the study was terminated on Day 42, colon was collected fordetermination of length and weight and histological analysis.Statistical analysis of end body weight and end colon weight and lengthof variant ICOSL-IgV Fc treated group to vehicle group was assessedusing two-tailed Student's t-test. Stool scores and DAI scores werecompared using Wilcoxon's non-parametric T test.

DAI results are shown in Tables 36 (DAI) and FIG. 36A, colonmeasurements are shown in Table 37, and colon histology results areshown in Table 38 and FIG. 36B. As shown tested variant ICOSL IgV-Fc(N52H/N57Y/Q100R) exhibited significantly reduced development of colitisin this model, which is consistent with the utility of this dualCD28/ICOS antagonist to effectively treat inflammatory bowel disease(IBD).

TABLE 36 Disease Activity Score End % body End stool End DAI Treatmentweight +/− SD p value score +/− SD p value score +/− SD p value Colitisnot induced/Vehicle 112.3% +/− 3.9% 0.3 +/− 0.5 0.3 +/− 0.5 Vehicle 92.7% +/− 11.7% <0.0001{circumflex over ( )}  1.3 +/− 1.30.0016{circumflex over ( )}  3.2 +/− 2.6 0.0003{circumflex over ( )} Fccontrol, 300 μg  96.2% +/− 5.4%  0.2656* 1.4 +/− 1.4 0.8836* 2.5 +/− 1.7 0.6948* ICOSL IgV-Fc 109.6% +/− 4.0% <0.0001~ 0.1 +/− 0.3 0.0014~ 0.1+/− 0.3 <0.0001~ (N52H/N57Y/Q100R), 400 μg {circumflex over ( )}Comparedto Colitis not induced/Vehicle *Compared to Vehicle ~Compared to Fccontrol, 300 μg

TABLE 37 Colon Measurements End colon End colon End Colon weight lengthweight/ Treatment (mg) +/− SD p value (mm) +/− SD p value length +/− SDp value Colitis not induced/Vehicle 175.3 +/− 16.9 82.8 +/− 5.2 2.1 +/−0.2 Vehicle 323.1 +/− 78.0 <0.0001{circumflex over ( )}  77.7 +/− 7.50.0668{circumflex over ( )}  4.2 +/− 1.2 <0.0001{circumflex over ( )} Fc control, 300 μg 276.7 +/− 92.4 0.1602* 77.6 +/− 8.9 0.9911* 3.6 +/−1.2 0.1843* ICOSL IgV-Fc 160.8 +/− 24.4 0.0004~ 79.4 +/− 6.1 0.5738~ 2.0+/− 0.3 0.0003~ (N52H/N57Y/Q100R), 400 μg {circumflex over ( )}Comparedto Colitis not induced/Vehicle *Compared to Vehicle ~Compared to Fccontrol, 300 μg

TABLE 38 Histology Results Colon (swiss roll) Cage Total Group # Mouse #Mouse ID# Mucosa Submucosa Muscularis score Colitis not 1 1 1-1 0 0 00.0 induced/Vehicle 2 1-2 0 0 0 0.0 3 1-3 0 0 0 0.0 4 1-4 0 0 0 0.0 2 12-1 1 1 0 2.0 2 2-2 0 0 0 0.0 3 2-3 4 2-4 3 1 3-1 2 3-2 3 3-3 4 3-4Average 0.2 0.2 0.0 0.3 Std Dev 0.4 0.4 0.0 0.8 SEM 0.2 0.2 0.0 0.3Vehicle 4 1 4-1 3 3 1 7.0 2 4-2 1 1 0 2.0 3 4-3 2 2 0 4.0 4 4-4 2 2 04.0 5 1 5-1 2 3 0 5.0 2 5-2 2 2 0 4.0 3 5-3 4 5-4  6a 1 6a-1  2 6a-2  36a-3   6b 1 6b-1  Average 2.0 2.2 0.2 4.3 Std Dev 0.6 0.8 0.4 1.6 SEM0.3 0.3 0.2 0.7 T-test vs colitis not induced/Vehicle 0.0001 0.00020.3409 0.0003 Fc control,  6b 2 6b-2  2 1 0 3.0 300 μg 3 6b-3  3 3 1 7.0 6c 1 6c-1  3 3 0 6.0 2 6c-2  2 2 2 6.0 3 6c-3  3 2 1 6.0 7 1 7-1 2 7-22 2 1 5.0 3 7-3 2 1 0 3.0 4 7-4 2 3 0 5.0 8 1 8-1 2 1 0 3.0 2 8-2 2 1 03.0 3 8-3 4 8-4 1 1 0 2.0 9 1 9-1 2 9-2 3 9-3 4 9-4 10  1 10-1  2 10-2 3 10-3  4 10-4  Average 2.2 1.8 0.5 4.5 Std Dev 0.6 0.9 0.7 1.7 SEM 0.20.3 0.2 0.5 T-test vs Vehicle 0.5676 0.4240 0.3663 0.8885 ICOSL IgV -Fc14  1 14-1  0 0 0 0.0 (N52H/N57Y/Q100R), 2 14-2  0 0 0 0.0 400 μg 314-3  0 0 0 0.0 4 14-4  0 0 0 0.0 15  1 15-1  0 0 0 0.0 2 15-2  0 0 00.0 3 15-3  4 15-4  16  1 16-1  2 16-2  3 16-3  4 16-4  Average 0.0 0.00.0 0.0 Std Dev 0.0 0.0 0.0 0.0 SEM 0.0 0.0 0.0 0.0 T-test vs Fc control0.0000 0.0002 0.1315 0.0000

The present invention is not intended to be limited in scope to theparticular disclosed embodiments, which are provided, for example, toillustrate various aspects of the invention. Various modifications tothe compositions and methods described will become apparent from thedescription and teachings herein. Such variations may be practicedwithout departing from the true scope and spirit of the disclosure andare intended to fall within the scope of the present disclosure.

What is claimed:
 1. A variant ICOS Ligand (ICOSL) polypeptide,comprising one or more amino acid modifications in an immunoglobulinsuperfamily (IgSF) domain of an ICOSL reference polypeptide, wherein theICOSL reference polypeptide is a truncated extracellular domaincomprising a contiguous sequence of amino acids comprising amino acids1-112 and a C-terminal truncation of at least 25 amino acids withreference to the ICOSL extracellular domain sequence set forth in SEQ IDNO:32.
 2. The variant ICOSL polypeptide of claim 1, wherein the variantICOSL polypeptide exhibits increased binding to the ectodomain(s) ofICOS or CD28 compared to the binding of the ICOSL reference polypeptidefor the same ectodomain(s).
 3. The variant ICOSL polypeptide of claim 1or claim 2, wherein the C-terminal truncation is of at least 30, atleast 40, at least 50, at least 60, at least 70, at least 80, at least90, at least 100, at least 125 amino acid residues.
 4. The variant ICOSLpolypeptide of any of claims 1-3, wherein the ICOSL referencepolypeptide is altered in or lacks a protease cleavage site set forth asamino acids 204-209 of SEQ ID NO:32.
 5. The variant ICOSL polypeptide ofany of claims 1-4, wherein the ICOSL reference polypeptide comprises thesequence of amino acids set forth in SEQ ID NO:545.
 6. The variant ICOSLpolypeptide of any of claims 1-4, wherein the ICOSL referencepolypeptide consists of the sequence of amino acids set forth in SEQ IDNO:545.
 7. A variant ICOSL Ligand (ICOSL) polypeptide, comprising one ormore amino acid modifications in an ICOSL reference polypeptide, whereinthe ICOSL reference polypeptide consists of the sequence of amino acidsset forth in SEQ ID NO:545.
 8. A variant ICOS Ligand (ICOSL)polypeptide, comprising one or more amino acid modifications in animmunoglobulin superfamily (IgSF) domain of an ICOSL referencepolypeptide, wherein the ICOSL reference polypeptide is altered in oneor more amino acids corresponding to amino acids 204-209 with referenceto SEQ ID NO:32.
 9. The variant ICOSL polypeptide of any of claims 1-8,wherein the one or more amino acid modifications are in a positioncorresponding to position(s) 52, 57 or 100, with reference to numberingof SEQ ID NO:32.
 10. The variant ICOSL polypeptide of any of claims 1-9,wherein the one or more amino acid modifications are selected from N52A,N52C, N52D, N52G, N52H, N52K, N52L, N52M, N52Q, N52R, N52S, N52T, N52V,N52Y, N52K, N57A, N57D, N57E, N57F, N57H, N57K, N57L, N57M, N57P, N57Q,N57S, N57T, N57V, N57Y, N57W, Q100A, Q100D, Q100G, Q100K, Q100L, Q100M,Q100N, Q100P, Q100R, Q100S, Q100T or Q100V, with reference to numberingof SEQ ID NO:32.
 11. The variant ICOSL polypeptide of any of claims1-10, wherein the one or more amino acid modifications are selected fromamong N52Y/N57Y/F138L/L203P, N52H/N57Y/Q100P, N52S/Y146C/Y152C,N52H/C198R, N52H/C140D/T225A, N52H/C198R/T225A, N52H/K92R, N52H/S99G,N57Y/Q100P, N52S/S130G/Y152C, N52S/Y152C, N52S/C198R, N52Y/N57Y/Y152C,N52Y/N57Y/H129P/C198R, N52H/L161P/C198R, N52S/T113E, N52D/S54P,N52K/L208P, N52S/Y152H, N52D/V151A, N52H/1143T, N52S/L80P,N52S/R75Q/L203P, N52S/D158G, N52D/Q133H, N52S/N57Y/H94D/L96F/L98F/Q100R,N52S/N57Y/H94D/L96F/L98F/Q100R/G103E/F120S, N52S/G103E, N52H/F78L/Q100R,N52H/N57Y/Q100R/V110D, N52H/N57Y/R75Q/Q100R/V110D, N52H/N57Y/Q100R,N52H/N57Y/L74Q/Q100R/V10D, N52H/Q100R, N52H/S121G,A20V/N52H/N57Y/Q100R/S109G, N52H/N57Y/R61S/Q100R/V110D/L173S,N52H/N57Y/Q100R/V122A, N52H/N57Y/Q100R/F172S, N52H/N57Y, N52S/F120S,N52S/V97A, N52S/G72R, N52S/A71T/A117T, N52S/E220G,Y47H/N52S/V107A/F120S, N52H/N57Y/Q100R/V110D/S132F/M175T,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R,Q37R/N52H/N57Y/Q100R/V110N/S142F/C198R/D217V/R221G,N52H/N57Y/Q100R/V110D/C198R,N52H/N57Y/Q100R/V1D/V16A/L161M/F172S/S192G/C198R, F27S/N52H/N57Y/V11N,N52S/H94E/L96I/S109N/L166Q, S18R/N52S/F93L/I143V/R221G,A20T/N52D/Y146C/Q164L,V11E/N30D/N52H/N57Y/H94E/L96I/L98F/N194D/V210A/I218T,N52S/H94E/L96I/V122M, N52H/N57Y/H94E/L96I/F120I/S126T/W153R/I218N,M10V/S18R/N30D/N52S/S126R/T139S/L203F, S25G/N30D/N52S/F120S/N227K,N30D/N52S/L67P/Q100K/D217G/R221K/T225S,N52H/N57Y/Q100R/V110D/A117T/T190S/C198R,N52H/N57Y/Q100R/V110D/F172S/C198R,S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173S/C198R,N52H/N57Y/V110A/C198R/R221I,M10I/S13G/N52H/N57Y/D77G/V110A/H129P/I143V/F172S/V193M, C198R,N52H/N57Y/R61C/Y62F/Q100R/V110N/F120S/C198R,N52H/N57Y/Q100R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/N144D/F172S/C198R, N52S/H94E/L98F/Q100R,N52S/E90A, N30D/K42E/N52S, N52S/F120S/I143V/I224V,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/S54P, T38P/N52S/N57D,N52H/C140del/T225A, N52H/F78L/Q100R/C198R, N52H/N57Y/R75Q/Q100P/V110D,N52H/N57Y/L74Q/V110D/S192G, N52H/S121G/C198R, N52S/F120S/N227K,N52S/A71T/A117T/T190A/C198R, T43A/N52H/N57Y/L74Q/D89G/V110D/F172S,N52H/N57Y/Q100R/V110D/S132F/M175T, N52D,N52H/N57Y/Q100R/V107I/V110D/I154F/C198R/R221G, N52Q/N207Q, N168Q/N207Q,N52Q/N168Q, N52Q/N84Q, N52Q/N119Q, N52Q/N84Q/N168Q, N52Q/N84Q/N207Q,N52Q/N119Q/N155Q, N52H/N84Q/N119Q, N52H/N84Q, N52H/N84Q/N168Q/N207Q,N52Q/N84Q/N155Q/N168Q, N52Q/N84Q/N119Q/N168Q, N52Q/N84Q/N119Q/N207Q,N52Q/N84Q/N119Q/N155Q, N52Q/N84Q/N119Q/N155Q/N207Q, N52Y/F138L/L203P,N57Y/Q100R/C198R, N57Y/F138L/L203P, Q100R/F138L,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R,N52H/N57Y/Q100R/L102R/H115R/F172S/C198R, N52H/V122A/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/N194D, N52H/N57Y/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/H115R, N52H/N57Y/Q100R/H115R,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/F172S, N52H/Q100R/H115R/I143T/F172S,N52H/N57Y/Q100P/H115R/F172S, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/E90A/H115R, N30D/K42E N52S/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N52S/H115R/F120S/I143V/C198R, N52S/H115R/F172S/C198R,N52H/N57Y/Q100P/C198R, N52H/N57Y/Q100P H115R/F172S/C198R,N52H/N57Y/Q100P/F172S/C198R, N52H/N57Y/Q100P/H115R,N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R, N52H/Q100R/H115R/F172S,N52H/Q100R/F172S/C198R, N52H/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/F172S/C198R, N52A/N57F/Q100S, N52A/N57H/Q100S,N52A/N57Y/Q100A, N52D/N57A/Q100A, N52D/Q100S, N52G/Q100A, N52H/Q100A,N52M/N57H/Q100S, N52M/N57W/Q100P, N52Q/N57F, N52Q/N57S/Q100A,N52R/N57L/Q100A, N52R/N57Y/Q100P, N52R/N57Y/Q100S, N52S/N57A/Q100A,N52S/N57H/Q100E, N52S/N57L/Q100S, N52S/N57M/Q100S, N52S/N57Y/Q100S,N52S/N57Y/Q100M, N52S/N57Y/Q100V, N52T/N57H/Q100S, N52T/N57H/Q100A,N52T/N57Y/Q100A, N52V/N57L/Q100A, N52H/N57Y/Q100K, N52K/N57Y/Q100R,N52L/N57H/Q100R, N52R/N57F/Q100N, N52R/N57F/Q100P, N52R/N57F/Q100R,N52R/N57F/Q100T, N52R/N57H/Q100K, N52R/N57L/Q100S, N52R/N57W/Q100K,N52R/N57W, N52R/N57Y/Q100R, N52C/N57E/Q100S, N52G/N57P/Q100D,N52G/N57V/Q100G, N52G/N57V, N52L/N57V, N52P/N57P, N52P/N57S/Q100G,N52S/N57L/Q100G, N52T/N57K/Q100P, N52V/N57T/Q100L, N57Q/Q100P, orR26S/N52H/N57Y/V110D/T137A/C198R, with reference to numbering of SEQ IDNO:32.
 12. The variant ICOSL polypeptide of any of claims 1-11, whereinthe one or more amino acid modifications are selected from amongN52A/N57Y/Q100A, N52D/Q100S, N52G/Q100A, N52M/N57H/Q100S,N52M/N57W/Q100P, N52Q/N57S/Q100A, N52R/N57L/Q100A, N52S/N57H/Q100E,N52S/N57L/Q100S, N52S/N57M/Q100S, N52S/N57Y/Q100M, N52T/N57H/Q100S,N52R/N57F/Q100P, N52R/N57F/Q100T, N52R/N57W/Q100K, N52R/N57W, N52G/N57V,N52L/N57V, N52S/N57L/Q100G, N52T/N57K/Q100P, N52S, N52H, N52D,N52Y/N57Y/F138L/L203P, N52H/N57Y/Q100P, N52S/Y146C/Y152C, N52H/C198R,N52H/C198R/T225A, N52H/K92R, N57Y, N52S/C198R, N52S/T113E, S54A,N52D/S54P, N52K/L208P, N52H/I143T, N52S/D158G, N52D/Q133H,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/V122A,N52H/N57Y/Q100R/F172S, N52H/N57Y/Q100R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/E90A,N52S/F120S/I143V/I224V, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/H115R/F120S/I143V/C198R, N52H/N57Y/Q100P/C198R,N52H/N57Y/Q100P/H115R/F172S/C198R, N52H/N57Y/Q100P/F172S/C198R,N52H/N57Y/Q100P/H115R, N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R,N52H/Q100R/H115R/F172S, N52H/Q100R/H115X/F172S/C198R,N52H/Q100R/H115R/F172S/C198R, N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S, N52H/N57Y/Q100R/H115R/F172S/C198R, Q100R,N52Y/F138L/L203P, N57Y/Q100R/C198R, N57Y/F138L/L203P, N52H, N57Y,N57Y/Q100P, Q100R/F138L, N52H/N57Y/Q100R/H115R, N52H/N57Y/Q100R/F172S,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R, N52H/N57Y/Q100R/L102RH115R/F172S/C198R, N52H/N57Y/Q100R/H115R F172S/N194D,N52H/N57Y/H115R/F172S/C198R, N52H/N57Y/Q100R/H115R/C198R,N52H/N57Y/H115R, N52H/Q100R/H115R/I143T/F172S,N52H/N57Y/Q100P/H115R/F172S, E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,N52S/E90A/H115R, N52S/E90A/H115R, or N30D/K42E/N52S/H115R, withreference to numbering of SEQ ID NO:32.
 13. The variant ICOSLpolypeptide of any of claims 1-12, comprising one or more amino acidmodifications N52H/Q100R.
 14. The variant ICOSL polypeptide of claim 13,wherein the variant ICOSL polypeptide has the sequence set forth in SEQID NO:567.
 15. The variant ICOSL polypeptide of any of claims 1-13,comprising one or more amino acid modifications are N52H/N57Y/Q100R. 16.The variant ICOSL polypeptide of claim 15, wherein the polypeptide hasthe sequence set forth in SEQ ID NO:565.
 17. The variant ICOSLpolypeptide of any of claims 1-12, comprising one or more amino acidmodifications are N52L/N57H/Q100R.
 18. The variant ICOSL polypeptide ofclaim 17, wherein the polypeptide has the sequence set forth in SEQ IDNO:761.
 19. The variant ICOSL polypeptide of any of claims 1-12,comprising the amino acid modification is N52D.
 20. The variant ICOSLpolypeptide of claim 19, wherein the polypeptide has the sequence setforth in SEQ ID NO:548.
 21. The variant ICOSL polypeptide of any ofclaims 8-12, wherein: the alteration comprises a deletion of one or morecontiguous amino acids corresponding to amino acids 204-209 withreference to SEQ ID NO:32; or the alteration comprises at least oneamino acid substitution at one or both of position 207 and 208corresponding to positions set forth in SEQ ID NO:32.
 22. The variantICOSL polypeptide of claim 21, wherein the at least one amino acidsubstitution is N207A, N207G or L208G with reference to numbering of SEQID NO:32, or a conservative amino acid substitution thereof.
 23. Thevariant ICOSL polypeptide of any of claims 1-22, wherein the variantICOSL polypeptide exhibits reduced proteolytic cleavage when expressedfrom a cell compared to a full-length extracellular domain of thevariant ICOSL polypeptide when expressed from the same cell.
 24. Thevariant ICOSL polypeptide of any of claims 1-8 and 21-23, wherein theone or more amino acid modifications are in a position corresponding toposition(s) selected from 10, 11, 13, 16, 18, 20, 25, 26, 27, 30, 33,37, 38, 42, 43, 47, 52, 54, 57, 61, 62, 67, 71, 72, 74, 75, 77, 78, 80,84, 89, 90, 92, 93, 94, 96, 97, 98, 99, 100, 102, 103, 107, 109, 110,111, 113, 115, 116, 117, 119, 120, 121, 122, 126, 129, 130, 132, 133,135, 137, 138, 139, 140, 142, 143, 144, 146, 151, 152, 153, 154, 155,156, 158, 161, 164, 166, 168, 172, 173, 175, 190, 192, 193, 194, 198,201, 203, 207, 208, 210, 212, 217, 218, 220, 221, 224, 225, or 227 withreference to numbering of SEQ ID NO:32.
 25. The variant ICOSLpolypeptide of any of claims 1-8 and 21-24, wherein the one or moreamino acid modifications are selected from M10V, M10I, V11E, S13G, E16V,S18R, A20T, A20V, S25G, R26S, F27C, F27S, N30D, Y33del, Q37R, T38P,K42E, T43A, Y47H, N52A, N52C, N52D, N52G, N52H, N52K, N52L, N52M, N52P,N52Q, N52R, N52S, N52T, N52V, N52Y, S54A, S54F, S54P, N57A, N57D, N57E,N57F, N57H, N57K, N57L, N57M, N57P, N57Q, N57S, N57T, N57V, N57W, N57Y,R61C, R61S, Y62F, L67P, A71T, G72R, L74Q, R75Q, D77G, F78L, L80P, N84Q,D89G, E90A, K92R, F93L, H94D, H94E, L96F, L96I, V97A, L98F, S99G, Q100A,Q100D, Q100E, Q100G, Q100K, Q100L, Q100M, Q100N, Q100P, Q100R, Q100S,Q100T, Q100V, L102R, G103E, V107A, V107I, S109G, S109N, V110A, V110D,V110N, E111del, T113E, H115Q, H115R, V116A, A117T, N119Q, F120I, F120S,S121G, V122A, V122M, S126R, S126T, H129P, S130G, S132F, Q133H, E135K,T137A, F138L, T139S, C140del, C140D, S142F, I143T, I143V, N144D, Y146C,V151A, Y152C, Y152H, W153R, I154F, N155H, N155Q, K156M, D158G, L161M,L161P, Q164L, L166Q, N168Q, F172S, L173S, M175T, T190A, T190S, S192G,V193A, V193M, N194D, C198R, N201S, L203F, L203P, N207Q, L208P, V210A,S212G, D217G, D217V, I218N, I218T, E220G, R221G, R221I, R221K, I224V,T225A, T225S, N227K with reference to numbering of SEQ ID NO:32, or aconservative amino acid substitution thereof.
 26. The variant ICOSLpolypeptide of any of claims 1-8 and 21-25, wherein the one or moreamino acid modifications are selected from among F120S/Y152H/N201S,E111del, Y33del, N168Q/N207Q, N84Q/N207Q, N155Q/N207Q, N119Q/N168Q,N119Q/N207Q, N119Q/N155Q, N84Q/N119Q, N84Q/N155Q/N168Q,N84Q/N168Q/N207Q, N84Q/N155H/N207Q, N155Q/N168Q/N207Q, N119QN155Q/N168Q, N119Q/N168Q/N207Q, N84Q/N119Q/N207Q, N119Q/N155H/N207Q,N84Q/N119Q/N155Q, N84Q/N119Q/N155Q/N168Q, N84Q/N155Q/N168Q/N207Q,N84Q/N119Q/N155Q/N207Q, N84Q/N119Q/N155Q/N168Q/N207Q or F138L/L203P,with reference to numbering of SEQ ID NO:32.
 27. The variant ICOSLpolypeptide of any of claims 1-8 and 21-25, wherein the one or moreamino acid modifications are selected from amino acid modificationsN52Y/N57Y/F138L/L203P, N52H/N57Y/Q100P, N52S/Y146C/Y152C, N52H/C198R,N52H/C140D/T225A, N52H/C198R/T225A, N52H/K92R, N52H/S99G, N57Y/Q100P,N52S/S130G/Y152C, N52S/Y152C, N52S/C198R, N52Y/N57Y/Y152C,N52Y/N57Y/H129P/C198R, N52H/L161P/C198R, N52S/T113E, N52D/S54P,N52K/L208P, N52S/Y152H, N52D/V151A, N52H/I143T, N52S/L80P,F120S/Y152H/N201S, N52S/R75Q/L203P, N52S/D158G, N52D/Q133H,N52S/N57Y/H94D/L96F/L98F/Q100R,N52S/N57Y/H94D/L96F/L98F/Q100R/G103E/F120S, N52S/G103E, N52H/F78L/Q100R,N52H/N57Y/Q100R/V110D, N52H/N57Y/R75Q/Q100R/V110D, N52H/N57Y/Q100R,N52H/N57Y/L74Q/Q100R/V110D, N52H/Q100R, N52H/S121G,A20V/N52H/N57Y/Q100R/S109G, N52H/N57Y/R61S/Q100R/V110D/L173S,N52H/N57Y/Q100R/V122A, N52H/N57Y/Q100R/F172S, N52H/N57Y, N52S/F120S,N52S/V97A, N52S/G72R, N52S/A71T/A117T, N52S/E220G,Y47H/N52S/V107A/F120S, N52H/N57Y/Q100R/V110D/S132F/M175T,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R,Q37R/N52H/N57Y/Q100R/V110N/S142F/C198R/D217V/R221G,N52H/N57Y/Q100R/V110D/C198R,N52H/N57Y/Q100R/V110D/V116A/L161M/F172S/S192G/C198R,F27S/N52H/N57Y/V110N, N52S/H94E/L96I/S109N/L166Q,S18R/N52S/F93L/I143V/R221G, A20T/N52D/Y146C/Q164L,V11E/N30D/N52H/N57Y/H94E/L96I/L98F/N194D/V210A/I218T,N52S/H94E/L96I/V122M, N52H/N57Y/H94E/L96I/F120I/S126T/W153R/I218N,M10V/S18R/N30D/N52S/S126R/T139S/L203F, S25G/N30D/N52S/F120S/N227K,N30D/N52S/L67P/Q100K/D217G/R221K/T225S,N52H/N57Y/Q100R/V110D/A117T/T190S/C198R,N52H/N57Y/Q100R/V110D/F172S/C198R,S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173S/C198R,N52H/N57Y/V110A/C198R/R221I,M1I/S13G/N52H/N57Y/D77G/V110A/H129P/I143V/F172S/V193M, C198R,N52H/N57Y/R61C/Y62F/Q100R/V110N/F120S/C198R,N52H/N57Y/Q100R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/N144D/F172S/C198R, N52S/H94E/L98F/Q100R,N52S/E90A, N30D/K42E/N52S, N52S/F120S/I143V/I224V,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/S54P, T38P/N52S/N57D,N52H/C140del/T225A, N52H/F78L/Q100R/C198R, N52H/N57Y/R75Q/Q100P/V110D,N52H/N57Y/L74Q/V110D/S192G, N52H/S121G/C198R, N52S/F120S/N227K,N52S/A71T/A117T/T190A/C198R, T43A/N52H/N57Y/L74Q/D89G/V110D/F172S,N52H/N57Y/Q100R/V110D/S132F/M175T,N52H/N57Y/Q100R/V107I/V110D/I154F/C198R/R221G, N52Q/N207Q, N52Q/N168Q,N52Q/N84Q, N52Q/N119Q, N52Q/N84Q/N168Q, N52Q/N84Q/N207Q,N52Q/N119Q/N155Q, N52H/N84Q/N119Q, N52H/N84Q, N52H/N84Q/N168Q/N207Q,N52Q/N84Q/N155Q/N168Q, N52Q/N84Q/N119Q/N168Q, N52Q/N84Q/N119Q/N207Q,N52Q/N84Q/N119Q/N155Q, N52Q/N84Q/N119Q/N155Q/N207Q, N52Y/F138L/L203P,N57Y/Q100R/C198R, N57Y/F138L/L203P, Q100R/F138L,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R,N52H/N57Y/Q100R/L102R/H115R/F172S/C198R, N52H/V122A/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/N194D, N52H/N57Y/H115R/F172S/C198R,N52H/N57Y/H115R, N52H/N57Y/Q100R/H115R,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/F172S, N52H/Q100R/H115R/I143T/F172S,N52H/N57Y/Q100P/H115R/F172S, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/E90A/H115R, N30D/K42E N52S/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N52S/H115R/F120S/I143V/C198R, N52S/H115R/F172S/C198R,N52H/N57Y/Q100P/C198R, N52H/N57Y/Q100P/H115R/F172S/C198R,N52H/N57Y/Q100P/F172S/C198R, N52H/N57Y/Q100P/H115R,N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R, N52H/Q100R/H115R/F172S,N52H/Q100R/F172S/C198R, N52H/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/F172S/C198R, N52A/N57F/Q100S, N52A/N57H/Q100S,N52A/N57Y/Q100A, N52D/N57A/Q100A, N52D/Q100S, N52G/Q100A, N52H/Q100A,N52M/N57H/Q100S, N52M/N57W/Q100P, N52Q/N57F, N52Q/N57S/Q100A,N52R/N57L/Q100A, N52R/N57Y/Q100P, N52R/N57Y/Q100S, N52S/N57A/Q100A,N52S/N57H/Q100E, N52S/N57L/Q100S, N52S/N57M/Q100S, N52S/N57Y/Q100S,N52S/N57Y/Q100M, N52S/N57Y/Q100V, N52T/N57H/Q100S, N52T/N57H/Q100A,N52T/N57Y/Q100A, N52V/N57L/Q100A, N52H/N57Y/Q100K, N52K/N57Y/Q100R,N52L/N57H/Q100R, N52R/N57F/Q100N, N52R/N57F/Q100P, N52R/N57F/Q100R,N52R/N57F/Q100T, N52R/N57H/Q100K, N52R/N57L/Q100S, N52R/N57W/Q100K,N52R/N57W, N52R/N57Y/Q100R, N52C/N57E/Q100S, N52G/N57P/Q100D,N52G/N57V/Q100G, N52G/N57V, N52L/N57V, N52P/N57P, N52P/N57S/Q100G,N52S/N57L/Q100G, N52T/N57K/Q100P, N52V/N57T/Q100L, N57Q/Q100P, orR26S/N52H/N57Y/V110D/T137A/C198R, with reference to numbering of SEQ IDNO:32.
 28. A variant ICOS Ligand (ICOSL) polypeptide, comprising an IgVdomain or specific binding fragment thereof, an IgC domain or a specificbinding fragment thereof, or both, wherein the variant ICOSL polypeptidecomprises one or more amino acid modifications in an ICOSL referencepolypeptide or a specific binding fragment thereof corresponding toamino acid modifications are selected from N52A, N52C, N52G, N52K, N52L,N52M, N52R, N52T, N52V, N57A, N57E, N57F, N57H, N57K, N57L, N57M, N57P,N57Q, N57S, N57T, N57V, N57W, Q100A, Q100D, Q100G, Q100L, Q100M, Q100N,Q100S, Q100T or Q100V with reference to SEQ ID NO:32.
 29. The variantICOSL polypeptide of claim 28, wherein the one or more amino acidmodifications are selected from among N52A/N57F/Q100S, N52A,/N57H/Q100S, N52A/N57Y/Q100A, N52D/N57A/Q100A, N52D/Q100S, N52G/Q100A,N52H/Q100A, N52M/N57H/Q100S, N52M/N57W/Q100P, N52Q/N57F,N52Q/N57S/Q100A, N52R/N57L/Q100A, N52R/N57Y/Q100P, N52R/N57Y/Q100S,N52S/N57A/Q100A, N52S/N57H/Q100E, N52S/N57L/Q100S, N52S/N57M/Q100S,N52S/N57Y/Q100S, N52S/N57Y/Q100M, N52S/N57Y/Q100V, N52T/N57H/Q100S,N52T/N57H/Q100A, N52T/N57Y/Q100A, N52V/N57L/Q100A, N52H/N57Y/Q100K,N52K/N57Y/Q100R, N52L/N57H/Q100R, N52R/N57F/Q100N, N52R/N57F/Q100P,N52R/N57F/Q100R, N52R/N57F/Q100T, N52R/N57H/Q100K, N52R/N57L/Q100S,N52R/N57W/Q100K, N52R/N57W, N52R/N57Y/Q100R, N52C/N57E/Q100S,N52G/N57P/Q100D, N52G/N57V/Q100G, N52G/N57V, N52L/N57V, N52P/N57P,N52P/N57S/Q100G, N52S/N57L/Q100G, N52T/N57K/Q100P, N52V/N57T/Q100L orN57Q/Q100P.
 30. The variant ICOSL polypeptide of claim 28 or claim 29,wherein the ICOSL reference polypeptide comprises (i) the sequence ofamino acids set forth in SEQ ID NO:32, (ii) a sequence of amino acidsthat has at least 95% sequence identity to SEQ ID NO:32; or (iii) aportion of (i) or (ii) comprising an IgV domain or IgC domain orspecific binding fragments thereof or both.
 31. The variant ICOSLpolypeptide of any of claims 28-30, wherein the variant ICOSLpolypeptide comprises the IgV domain or a specific binding fragmentthereof.
 32. The variant ICOSL polypeptide of any of claims 28-31,wherein the IgV domain or specific binding fragment thereof is the onlyICOSL portion of the variant ICOSL polypeptide.
 33. The variant ICOSLpolypeptide of any of claims 28-32, wherein the ICOSL referencepolypeptide comprises the sequence of amino acids set forth in SEQ IDNO:545.
 34. The variant ICOSL polypeptide of any of claims 28-32,wherein the ICOSL reference polypeptide consists of the sequence ofamino acids set forth in SEQ ID NO:545.
 35. The variant ICOSLpolypeptide of any of claims 1-34, wherein the variant ICOSL polypeptideexhibits increased binding to the ectodomain(s) of ICOS or CD28 comparedto the binding of the ICOSL reference polypeptide for the sameectodomain(s).
 36. The variant ICOSL polypeptide of any of claims 1-34,wherein the variant ICOSL polypeptide exhibits increased binding to theectodomain(s) of ICOS and CD28 compared to the binding of the ICOSLreference polypeptide for the same ectodomain(s).
 37. The variant ICOSLpolypeptide of any of claims 2-6, 9-27, 35 and 36, wherein the bindingis increased more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold,40-fold, 50-fold or 60-fold.
 38. The variant ICOSL polypeptide of any ofclaims 2-6, 9-27, 35, 36 and 37, wherein the ICOS is a human ICOS. 39.The variant ICOSL polypeptide of any of claims 2-6, 9-27, 35, 36, 37 and38, wherein the CD28 is a human CD28.
 40. The variant ICOSL polypeptideof any of claims 1-39, wherein the variant ICOSL polypeptide comprisesup to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19or 20 amino acid modifications, optionally amino acid substitutions,insertions and/or deletions.
 41. The variant ICOSL polypeptide of any ofclaims 1-40 that is a soluble protein.
 42. The variant ICOSL polypeptideof any of claims 1-41, wherein: the variant ICOSL polypeptide lacks atransmembrane domain and intracellular signaling domain; and/or whenexpressed from a cell, the variant ICOSL polypeptide is not expressed onthe surface of the cell.
 43. The variant ICOSL polypeptide of any ofclaims 1-40, wherein the variant ICOSL polypeptide further comprises atransmembrane domain.
 44. The variant ICOSL polypeptide of claim 43,further comprising a cytoplasmic signaling domain linked to thetransmembrane domain.
 45. An immunomodulatory protein, comprising thevariant ICOSL polypeptide of any of claims 1-44 and a half-lifeextending moiety.
 46. The immunomodulatory protein of claim 45, whereinthe half-life extending moiety comprises a multimerization domain,albumin, an albumin-binding polypeptide, Pro/Ala/Ser (PAS), a C-terminalpeptide (CTP) of the beta subunit of human chorionic gonadotropin,polyethylene glycol (PEG), long unstructured hydrophilic sequences ofamino acids (XTEN), hydroxyethyl starch (HES), an albumin-binding smallmolecule, or a combination thereof.
 47. The immunomodulatory protein ofclaim 45 or claim 46, wherein the half-life extending moiety is orcomprises a multimerization domain.
 48. The immunomodulatory protein ofclaim 47, wherein the multimerization domain is or comprises an Fcregion of an immunoglobulin.
 49. The immunomodulatory protein of claim47 or claim 48, wherein the variant ICOSL polypeptide is linked,directly or indirectly via a linker, to the multimerization domain. 50.The immunomodulatory protein of any of claims 47-49, wherein theimmunomodulatory protein is a multimer comprising a first variant ICOSLpolypeptide linked to a first multimerization domain and a secondvariant ICOSL polypeptide linked to a second multimerization domain,wherein the first and second multimerization domains interact to form amultimer comprising the first and second variant ICOSL polypeptide. 51.The immunomodulatory protein of claim 50, wherein the multimer is adimer.
 52. The immunomodulatory protein of claim 50 or claim 51, whereinthe first variant ICOSL polypeptide and the second variant ICOSLpolypeptide are the same.
 53. The immunomodulatory protein of claim 51or claim 52, wherein the dimer is a homodimer.
 54. The immunomodulatoryprotein of claim 51, wherein the dimer is a heterodimer.
 55. Theimmunomodulatory protein of any of claims 48-54, wherein the Fc regionis a human IgG or is a variant Fc region comprising one or more aminoacid substitutions compared to the wildtype human IgG1.
 56. Theimmunomodulatory protein of any of claims 48-55, wherein the Fc regioncomprises the sequence of amino acids set forth in SEQ ID NO: 226 or avariant thereof that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% sequence identity to SEQ ID NO:226.
 57. Theimmunomodulatory protein of any of claims 48-56, wherein the Fc regionexhibits one or more effector functions.
 58. The immunomodulatoryprotein of any of claims 48-56, wherein the Fc region is a variant Fcregion that exhibits one or more reduced effector function compared toan Fc of a wildtype human IgG1.
 59. The immunomodulatory protein ofclaim 58, wherein the variant Fc region comprises one or more amino acidsubstitutions selected from N297G, E233P/L234V/L235A/G236del/S267K orL234A/L235E/G237A, wherein the residue is numbered according to the EUindex of Kabat.
 60. The immunomodulatory protein of claim 59, whereinthe variant Fc region further comprises the amino acid substitutionC220S, wherein the residues are numbered according to the EU index ofKabat.
 61. The immunomodulatory protein of claim 59 or claim 60, whereinthe Fc region comprises K447del, wherein the residue is numberedaccording to the EU index of Kabat.
 62. The immunomodulatory protein ofany of claims 59-61, wherein the Fc region comprises the sequence ofamino acids set forth in SEQ ID NO:476 or SEQ ID NO:632.
 63. Theimmunomodulatory protein of any of claims 59-61, wherein the Fc regioncomprises the sequence of amino acids set forth in SEQ ID NO:478 or SEQID NO:634.
 64. The immunomodulatory protein of any of claims 59-61,wherein the Fc region comprises the sequence of amino acids set forth inSEQ ID NO:477.
 65. The immunomodulatory protein of any of claims 59-61,wherein the Fc region comprises the sequence of amino acids set forth inSEQ ID NO:633.
 66. The immunomodulatory protein of any of claims 59-61,wherein the Fc region comprises the sequence of amino acids set forth inSEQ ID NO:474.
 67. The immunomodulatory protein of any of claims 59-61,wherein the Fc region comprises the sequence of amino acids set forth inSEQ ID NO:637.
 68. The immunomodulatory protein of any of claims 49-67,wherein the variant ICOSL polypeptide is linked via a linker to the Fcregion.
 69. The immunomodulatory protein of any of claims 49-68, whereinthe linker comprises 1 to 10 amino acids.
 70. The immunomodulatoryprotein of any of claims 49-69, wherein the linker is AAA.
 71. Theimmunomodulatory protein of any of claims 49-69, wherein the linker isG4S (SEQ ID NO:636).
 72. The immunomodulatory protein of any of claims49-69, wherein the linker is (G₄S)₂ (SEQ ID NO:229).
 73. Theimmunomodulatory protein of any of claims 49-69, wherein the linker isGSGGGGS linker (SEQ ID NO: 635).
 74. An immunomodulatory protein,comprising the variant ICOSL polypeptide of any of claims 1-44 linked toa second polypeptide comprising an immunoglobulin superfamily (IgSF)domain.
 75. The immunomodulatory polypeptide of claim 74, wherein theIgSF domain of the second polypeptide exhibits increased binding to oneor more of its cognate binding partner(s) compared to the unmodified orwild-type IgSF domain.
 76. The immunomodulatory protein of claim 74 orclaim 75, wherein the variant ICOSL polypeptide is capable ofspecifically binding to CD28 or ICOS and the IgSF domain of the secondpolypeptide is capable of binding to a binding partner other than onespecifically bound by the ICOSL variant polypeptide.
 77. Theimmunomodulatory polypeptide of any of claims 74-76, wherein the IgSFdomain of the second polypeptide is a tumor-localizing moiety that bindsto a ligand expressed on a tumor.
 78. The immunomodulatory polypeptideof claim 77, wherein the ligand is B7H6.
 79. The immunomodulatorypolypeptide of claim 77 or claim 78, wherein the IgSF domain is fromNKp30.
 80. The immunomodulatory polypeptide of any of claims 74-79,wherein the IgSF domain of the second polypeptide is or comprises an IgVdomain.
 81. The immunomodulatory polypeptide of any of claims 74-80,wherein the IgSF domain of the second polypeptide has the sequence setforth in SEQ ID NO:504.
 82. The immunomodulatory polypeptide of any ofclaims 45-81, wherein the variant ICOSL polypeptide is or comprise anIgV domain.
 83. The immunomodulatory polypeptide of any of claims 45-82,wherein the variant ICOSL polypeptide comprises amino acid modificationsN52H/Q100R.
 84. The immunomodulatory polypeptide of claim 83, whereinthe variant ICOSL polypeptide has the sequence set forth in SEQ IDNO:567.
 85. The immunomodulatory polypeptide of any of claims 45-83,wherein the variant ICOSL polypeptide comprises amino acid modificationsN52H/N57Y/Q100R.
 86. The immunomodulatory polypeptide of claim 85,wherein the variant ICOSL polypeptide has the sequence set forth in SEQID NO:565.
 87. The immunomodulatory polypeptide of any of claims 45-82,wherein the variant ICOSL polypeptide comprises amino acid modificationsare N52L/N57H/Q100R.
 88. The immunomodulatory polypeptide of claim 87,wherein the variant ICOSL polypeptide has the sequence set forth in SEQID NO:761.
 89. The immunomodulatory polypeptide of any of claims 45-82,wherein the variant ICOSL polypeptide comprises the amino acidmodification is N52D.
 90. The immunomodulatory polypeptide of claim 89,wherein the variant ICOSL polypeptide has the sequence set forth in SEQID NO:548.
 91. The immunomodulatory protein of any of claims 74-90,wherein the immunomodulatory protein comprises a multimerization domainlinked to one or both of the variant ICOSL polypeptide or the secondpolypeptide comprising the IgSF domain.
 92. The immunomodulatory proteinof claim 91, wherein the multimerization domain is an Fc region.
 93. Theimmunomodulatory protein of any of claims 74-92 that is dimeric.
 94. Theimmunomodulatory protein of claim 93 that is homodimeric.
 95. Theimmunomodulatory protein of claim 93 that is heterodimeric.
 96. Theimmunomodulatory protein of any of claims 92-95, wherein the Fc regionis a human IgG or is a variant Fc region comprising one or more aminoacid substitutions compared to the wildtype human IgG1.
 97. Theimmunomodulatory protein of any of claims 92-96, wherein the Fc regioncomprises the sequence of amino acids set forth in SEQ ID NO: 226 or avariant thereof that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% sequence identity to SEQ ID NO:226.
 98. Theimmunomodulatory protein of any of claims 92-97 wherein the Fc regionexhibits one or more effector functions.
 99. The immunomodulatoryprotein of any of claims 92-97, wherein the Fc region is a variant Fcregion that exhibits one or more reduced effector function compared toan Fc of a wildtype human IgG1.
 100. The immunomodulatory protein ofclaim 99, wherein the variant Fc region comprises one or more amino acidsubstitutions selected from N297G, E233P/L234V/L235A/G236del/S267K orL234A/L235E/G237A, wherein the residue is numbered according to the EUindex of Kabat.
 101. The immunomodulatory protein of claim 100, whereinthe variant Fc region further comprises the amino acid substitutionC220S, wherein the residues are numbered according to the EU index ofKabat.
 102. The immunomodulatory protein of claim 100 or claim 101,wherein the Fc region comprises K447del, wherein the residue is numberedaccording to the EU index of Kabat.
 103. The immunomodulatory protein ofany of claims 100-102, wherein the Fc region comprises the sequence ofamino acids set forth in SEQ ID NO:476 or SEQ ID NO:632.
 104. Theimmunomodulatory protein of any of claims 100-102, wherein the Fc regioncomprises the sequence of amino acids set forth in SEQ ID NO:478 or SEQID NO:634.
 105. The immunomodulatory protein of any of claims 100-102,wherein the Fc region comprises the sequence of amino acids set forth inSEQ ID NO:477.
 106. The immunomodulatory protein of any of claims100-102, wherein the Fc region comprises the sequence of amino acids setforth in SEQ ID NO:633.
 107. The immunomodulatory protein of any ofclaims 100-102, wherein the Fc region comprises the sequence of aminoacids set forth in SEQ ID NO:474.
 108. The immunomodulatory protein ofany of claims 100-102, wherein the Fc region comprises the sequence ofamino acids set forth in SEQ ID NO:637.
 109. The immunomodulatoryprotein of any of claims 74-108, wherein the variant ICOSL polypeptideand the IgSF domain of the second polypeptide are linked by a linker.110. The immunomodulatory protein of claim 109, wherein the linker is3×GGGGS (SEQ ID NO: 228).
 111. The immunomodulatory protein of any ofclaims 91-110, wherein the multimerization domain is linked via a linkerto one or both of the variant ICOSL polypeptide or the secondpolypeptide comprising the IgSF domain.
 112. The immunomodulatoryprotein of claim 111, wherein the linker is GSGGGGS (SEQ ID NO: 635).113. A conjugate comprising the variant ICOSL polypeptide of any ofclaims 1-44 or immunomodulatory protein of any of claims 45-112 and aheterologous moiety.
 114. The conjugate of claim 113, wherein theconjugate is a fusion protein.
 115. The conjugate of claim 113 or claim114, wherein the moiety is a targeting moiety that specifically binds toa molecule on the surface of a cell.
 116. The conjugate of claim 115,wherein the targeting moiety specifically binds to a molecule on thesurface of an immune cell.
 117. The conjugate of claim 116, wherein theimmune cell is an antigen presenting cell or a lymphocyte.
 118. Theconjugate of claim 115, wherein the targeting moiety is atumor-localizing moiety that binds to a molecule on the surface of atumor.
 119. The conjugate of any of claims 115-118, wherein thetargeting moiety is an antibody or antigen-binding fragment.
 120. Theconjugate of claim 119, wherein the antibody is selected from cetuximab,panitumumab, zalutumumab, nimotuzumab, trastuzumab, Ado-trastuzumabemtansine, Tositumomab (Bexxar®), Rituximab (Rituxan, Mabthera),Ibritumomab tiuxetan (Zevalin), Daclizumab (Zenapax), Gemtuzumab(Mylotarg), Alemtuzumab, CEA-scan Fab fragment, OC125 monoclonalantibody, ab75705, B72.3, Bevacizumab (Avastin®), Afatinib, Axitinib,Bosutinib, Cabozantinib, Ceritinib, Crizotinib, Dabrafenib, Dasatinib,Dinutuximab, Erlotinib, Everolimus, Ibrutinib, Imatinib, Lapatinib,Lenvatinib, Nilotinib, Olaparib, Olaratumab, Palbociclib, Pazopanib,Pertuzumab, Ramucirumab, Regorafenib, Ruxolitinib, Sorafenib, Sunitinib,Temsirolimus, Trametinib, Vandetanib, Vemurafenib, Vismodegib,Basiliximab, Ipilimumab, Nivolumab, pembrolizumab, MPDL3280A,Pidilizumab (CT-011), AMP-224, MSB001078C, or MEDI4736, BMS-935559,LY3300054, atezolizumab, avelumab or durvalumab or is an antigen-bindingfragment thereof.
 121. A monovalent fusion protein comprising: (a) avariant ICOSL polypeptide of any of claims 1-44; and (b) a label fordetection or purification of the variant ICOSL polypeptide.
 122. Thefusion protein of claim 121, wherein the label for detection orpurification is selected from a poly-histidine (His) tag, a FLAG-tag, aMyc-tag, or a fluorescent protein-tag.
 123. A nucleic acid molecule(s),encoding a variant ICOSL polypeptide of any of claims 1-44, animmunomodulatory protein of any of claims 45-112 or a fusion protein ofany of claims 114-122.
 124. A vector, comprising the nucleic acidmolecule(s) of claim
 123. 125. A cell, comprising the nucleic acidmolecule(s) of claim 123 or the vector of claim
 124. 126. A method ofproducing an immunomodulatory protein comprising a variant ICOSLpolypeptide, comprising introducing the nucleic acid molecule of claim123 or vector of claim 124 into a host cell under conditions to expressthe protein in the cell.
 127. The method of claim 126 that is amammalian cell.
 128. The cell of claim 126 or claim 127 that is aChinese Hamster Ovary (CHO) cell or a derivative thereof.
 129. The cellof any of claims 126-128 that is CHO DG44.
 130. The method of any ofclaims 126-129, further comprising isolating or purifying the proteinfrom the cell.
 131. A protein produced by the method of any of claims126-130.
 132. A composition comprising a protein comprising a variantICOSL polypeptide of any of claims 1-44 or an immunomodulatory proteinof any of claims 45-112, wherein at least 95%, 96%, 97%, 98%, 99% of theindividual sequences of the protein or the immunomodulatory protein inthe composition have an identical sequence length.
 133. The compositionof claim 132, wherein the protein or immunomodulatory protein ispurified from Chinese Hamster Ovary Cells or a derivative thereof. 134.A polynucleotide comprising a nucleic acid encoding a variant ICOSLpolypeptide comprising a transmembrane domain of claim 43 or claim 44and one or more nucleic acid encoding one or more chain of a recombinantantigen receptor.
 135. The polynucleotide of claim 134, wherein therecombinant antigen receptor is a chimeric antigen receptor (CAR) or anengineered T cell receptor (TCR).
 136. The polynucleotide of claim 134or claim 135, wherein each of the nucleic acid encoding the variantICOSL polypeptide and the one or more nucleic acid encoding one or morechain of the recombinant receptor is separated by a nucleic acidencoding a self-cleaving peptide or a peptide that causes ribosomeskipping.
 137. An engineered cell comprising the variant ICOSLpolypeptide of any of claims 1-44, the immunomodulatory protein of anyof claims 45-112, or the fusion protein of any of claims 114-122. 138.The engineered cell of claim 137, wherein: the nucleic acid encoding thevariant ICOSL polypeptide, immunomodulatory protein or fusion proteinencodes a signal peptide; the variant ICOSL polypeptide,immunomodulatory protein or fusion protein does not comprise atransmembrane domain and/or is not expressed on the surface of the cell;and/or the variant ICOSL polypeptide, immunomodulatory protein or fusionprotein is secreted from the engineered cell.
 139. The engineered cellof claim 137, wherein the engineered cell comprises a variant ICOSLpolypeptide comprising a transmembrane domain of claim 43 or claim 44.140. The engineered cell of any of claims 137-139, wherein the cell isan immune cell.
 141. The engineered cell of claim 140, wherein theimmune cell is an antigen presenting cell (APC) or a lymphocyte. 142.The engineered cell of any of claims 137-141 that is a primary humancell.
 143. The engineered cell of any of claims 137-142, furthercomprising a chimeric antigen receptor (CAR) or an engineered T-cellreceptor.
 144. An infectious agent, comprising a nucleic acid moleculeencoding a variant ICOSL polypeptide of any of claims 1-44 or animmunomodulatory protein of any of claims the immunomodulatory proteinof any of claims 45-112, or the fusion protein of any of claims 114-122.145. The infectious agent of claim 144, wherein the infectious agent isa bacteria or a virus.
 146. A pharmaceutical composition, comprising thevariant ICOSL polypeptide of any of claims 1-44, the immunomodulatoryprotein of any of claims 45-112, a conjugate or fusion protein of any ofclaims 113-122 or an engineered cell of any of claims 137-143 or aninfectious agent of claim 144 or claim
 145. 147. The pharmaceuticalcomposition of claim 146, comprising a pharmaceutically acceptableexcipient.
 148. An article of manufacture comprising the pharmaceuticalcomposition of claim 146 or claim 147 in a vial.
 149. A kit comprisingthe composition of claim 146 or claim 147 or the article of manufactureof claim 148, and instructions for use.
 150. A method of modulating animmune response in a subject, comprising administering thepharmaceutical composition of claim 146 or claim 147 to the subject.151. A method of modulating an immune response in a subject, comprisingadministering the engineered cells of any of claims 137-143 to thesubject.
 152. The method of claim 151, wherein the engineered cells areautologous to the subject.
 153. The method of claim 151, wherein theengineered cells are allogenic to the subject.
 154. The method of any ofclaims 150-153, wherein modulating the immune response treats a diseaseor condition in the subject.
 155. A method of treating a disease orcondition in a subject, the method comprising administering thepharmaceutical composition of claim 146 or claim 147 to the subject.156. The method of any of claims 150-155, wherein the immune response isincreased in the subject.
 157. The method of any of claims 150 and154-156, wherein the pharmaceutical composition comprises animmunomodulatory protein or conjugate comprising a variant ICOSLpolypeptide linked to a tumor-localizing moiety.
 158. The method ofclaim 157, wherein the tumor-localizing moiety is or comprises a bindingmolecule that recognizes a tumor antigen.
 159. The method of any ofclaims 150 and 154-158, wherein the pharmaceutical composition comprisesthe immunomodulatory protein of any of claims 77-112 or the conjugate orfusion protein of any of claims 113-120 is administered to the subject.160. The method of any of claims 150-156, wherein the pharmaceuticalcomposition comprises an engineered cell comprising a variant ICOSLpolypeptide that is a transmembrane immunomodulatory protein of claim 43or claim
 44. 161. The method of any of claims 154-160, wherein thedisease or condition is a tumor or cancer.
 162. The method of any one ofclaims 154-161, wherein the disease or condition is selected frommelanoma, lung cancer, bladder cancer, a hematological malignancy, livercancer, brain cancer, renal cancer, breast cancer, pancreatic cancer,colorectal cancer, spleen cancer, prostate cancer, testicular cancer,ovarian cancer, uterine cancer, gastric carcinoma, a musculoskeletalcancer, a head and neck cancer, a gastrointestinal cancer, a germ cellcancer, or an endocrine and neuroendocrine cancer.
 163. The method ofany of claims 150-155, wherein the immune response is decreased. 164.The method of any of claims 150-155 and 163, wherein the pharmaceuticalcomposition comprises a variant ICOSL polypeptide immunomodulatoryprotein that is an immunomodulatory Fc fusion protein.
 165. The methodof any of claims 150-155, 163 and 164, wherein the pharmaceuticalcomposition comprises a variant ICOSL polypeptide of any of claims 1-42or an immunomodulatory protein of any of claims 45-76.
 166. The methodof any of claims 150-155 and 163, wherein the pharmaceutical compositioncomprises an engineered cell comprising a secretable variant ICOSLpolypeptide is administered to the subject.
 167. The method of any ofclaims 150-155, 163 and 166, wherein the pharmaceutical compositioncomprises an engineered cell of claim 137 or claim
 138. 168. The methodof any of claims 150-155 and 163, wherein the pharmaceutical compositioncomprises an infectious agent encoding a variant ICOSL polypeptide thatis a secretable immunomodulatory protein is administered to the subject,optionally under conditions in which the infectious agent infects atumor cell or immune cell and the secretable immunomodulatory protein issecreted from the infected cell.
 169. The method of any of claims154-155 and 163-168, wherein the disease or condition is an inflammatoryor autoimmune disease or condition.
 170. The method of any of claims154-155 and 163-169, wherein the disease or condition is anAntineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, avasculitis, an autoimmune skin disease, transplantation, a Rheumaticdisease, an inflammatory gastrointestinal disease, an inflammatory eyedisease, an inflammatory neurological disease, an inflammatory pulmonarydisease, an inflammatory endocrine disease, or an autoimmunehematological disease.
 171. The method of claim 169 or claim 170,wherein the disease or condition is selected from inflammatory boweldisease, transplant, Crohn's disease, ulcerative colitis, multiplesclerosis, asthma, rheumatoid arthritis, or psoriasis.